Neoantigens and uses thereof

ABSTRACT

Disclosed herein relates to immunotherapeutic compositions comprising immunotherapeutic peptides comprising neoepitopes, polynucleotides encoding the immunotherapeutic peptides, antigen presenting cells comprising the immunotherapeutic peptides or polynucleotides, or T cell receptors specific for the neoepitopes. Also disclosed herein is use of the immunotherapeutic compositions.

CROSS-REFERENCE

This application claims benefit of U.S. Provisional Application No.62/687,188, filed on Jun. 19, 2018 and 62/800,735, filed on Feb. 4,2019; which are incorporated herein by reference in their entirety.

BACKGROUND

Cancer immunotherapy is the use of the immune system to treat cancer.Immunotherapies exploit the fact that cancer cells often have moleculeson their surface that can be detected by the immune system, known astumor antigens, which are often proteins or other macromolecules (e.g.carbohydrates). Active immunotherapy directs the immune system to attacktumor cells by targeting tumor antigens. Passive immunotherapies enhanceexisting anti-tumor responses and include the use of monoclonalantibodies, lymphocytes and cytokines. Tumor vaccines are typicallycomposed of tumor antigens and immunostimulatory molecules (e.g.,adjuvants, cytokines or TLR ligands) that work together to induceantigen-specific cytotoxic T cells (CTLs) that recognize and lyse tumorcells. One of the critical barriers to developing curative andtumor-specific immunotherapy is the identification and selection ofhighly specific and restricted tumor antigens to avoid autoimmunity.

Tumor neoantigens, which arise as a result of genetic change (e.g.,inversions, translocations, deletions, missense mutations, splice sitemutations, etc.) within malignant cells, represent the mosttumor-specific class of antigens and can be patient-specific or shared.Tumor neoantigens are unique to the tumor cell as the mutation and itscorresponding protein are present only in the tumor. They also avoidcentral tolerance and are therefore more likely to be immunogenic.Therefore, tumor neoantigens provide an excellent target for immunerecognition including by both humoral and cellular immunity. However,tumor neoantigens have rarely been used in cancer vaccine or immunogeniccompositions due to technical difficulties in identifying them,selecting optimized antigens, and producing neoantigens for use in avaccine or immunogenic composition. Accordingly, there is still a needfor developing additional cancer therapeutics.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

SUMMARY

In some aspects, provided herein is a composition comprising at leastone polypeptide comprising two or more mutant RAS peptide sequencesselected from the group consisting of KLVVVGADGV, KLVVVGACGV,KLVVVGAVGV, LVVVGADGV, LVVVGACGV, LVVVGAVGV; GADGVGKSAL, GACGVGKSAL,GAVGVGKSAL, GADGVGKSA, GACGVGKSA, GAVGVGKSA; and/or VVGADGVGK,VVGACGVGK, VVGAVGVGK, VVVGADGVGK, VVVGACGVGK, VVVGAVGVGK; at least onepolynucleotide encoding the at least one polypeptide.

In some embodiments, the composition comprises a mixture of the three ormore mutant RAS peptide sequences.

In some aspects, provided herein is a composition comprising: at leastone polypeptide comprising two or more mutant RAS peptide sequences eachcomprising: at least 8 contiguous amino acids of a mutant RAS proteincomprising a mutation at G12, and the mutation at G12; and furtherwherein three or more amino acid residues that are heterologous to themutant RAS protein are linked to the N-terminus or C-terminus of the twoor more mutant RAS peptide sequences, wherein the three or more aminoacid residues enhance processing of the mutant RAS peptide sequences incell and/or enhance presentation of an epitope of the mutant RAS peptidesequences; or at least one polynucleotide encoding the at least onepolypeptide.

In some embodiments, the three or more amino acid residues that areheterologous to the mutant RAS protein are linked to the N-terminus orC-terminus of the two or more mutant RAS peptide sequences comprises anamino acid sequence of a protein of CMV such as pp65, HIV, or MART-1.

In some embodiments, the three or more amino acid residues that areheterologous to the mutant RAS protein are linked to the N-terminus orC-terminus of the two or more mutant RAS peptide sequences comprises atleast 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, or 40 amino acids.

In some embodiments, the three or more amino acid residues that areheterologous to the mutant RAS protein are linked to the N-terminus orC-terminus of the two or more mutant RAS peptide sequences comprises atmost 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,50, 60, 70, 80, 90, or 100 amino acids.

In some aspects, provided herein is a composition comprising at leastone polypeptide of the formula(Xaa_(N))_(N)-(Xaa_(RAS))_(P)-(Xaa_(C))_(C) wherein P is an integergreater than 7; (Xaa_(RAS))_(P) is a mutant RAS peptide sequencecomprising at least 8 contiguous amino acids of a mutant RAS protein;the at least 8 contiguous amino acids comprising at least 8 contiguousamino acids of the sequence Lys1 Leu2 Val3 Val4 Val5 Gly6 Ala7 Xaa8 Gly9Val10 Gly11 Lys12 Ser13 Ala14 Leu15 N is (i) 0 or (ii) an integergreater than 2; (Xaa_(N))_(N) is any amino acid sequence heterologous tothe mutant RAS protein; C is (i) 0 or (ii) an integer greater than 2;(Xaa_(C))_(C) is any amino acid sequence heterologous to the mutant RASprotein; Xaa8 is selected from the group consisting of Asp, Val, Cys,Ala, Arg and Ser; the polypeptide is not KLVVVGAVGVGKSALTIQL; and both Nand C are not 0; or at least one polynucleotide encoding the at leastone polypeptide.

In some embodiments, (Xaa)_(N) and/or (XaaC)_(C) comprises an amino acidsequence of a protein of CMV such as pp65, HIV, or MART-1.

In some embodiments, N and/or C is an integer greater than 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.

In some embodiments, N and/or C is an integer less than 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90,or 100.

In some embodiments, N is 0.

In some embodiments, C is 0.

In some aspects, provided herein is a composition comprising at leastone polypeptide comprising of an amino acid sequence ofXaa₁-Xaa₂-Val₃-Val₄-Val₅-Gly₆-Ala₇-Xaa₈-Gly₉-Xaa₁₀ wherein Xaa₁ is notAla; with the proviso that when Xaa₁ is not Lys, Xaa₂ is Leu and/orXaa₁₀ is Gly; Xaa₂ is not Glu; with the proviso that when Xaa₂ is notLeu, Xaa₁ is Lys and/or Xaa₁₀ is Gly; Xaa₈ is selected from the groupconsisting of Asp, Val, Cys, Ala, Arg and Ser; with the proviso thatwhen Xaa₈ is Glu, Xaa1 is not Tyr and/or Xaa₂ is not Leu, and with theproviso that when Xaa₈ is Val, Xaa₁ is not Lys; Xaa₁₀ is any amino acid;with the proviso that when Xaa₁₀ is not Gly, Xaa₁ is Lys and/or Xaa₂ isLeu; and the polypeptide comprises an HLA-A02:01-restricted T cellepitope, HLA-A03:01-restricted T cell epitope, an HLA-A11:01-restrictedT cell epitope, an HLA-A03:02-restricted T cell epitope, anHLA-A30:01-restricted T cell epitope, an HLA-A31:01-restricted T cellepitope, an HLA-A33:01-restricted T cell epitope, anHLA-A33:03-restricted T cell epitope, an HLA-A68:01-restricted T cellepitope, or an HLA-A74:01-restricted T cell epitope that binds to anHLA-A02:01, HLA-A03:01, HLA-A11:01, HLA-A03:02, HLA-A30:01, HLA-A31:01,HLA-A33:01, HLA-A33:03, HLA-A68:01, and/or an HLA-A74:01 molecule; andinduces an HLA-A02:01-restricted cytotoxic T cell response, anHLA-A02:01-restricted cytotoxic T cell response, HLA-A03:01-restrictedcytotoxic T cell response, an HLA-A11:01-restricted cytotoxic T cellresponse, an HLA-A03:02-restricted cytotoxic T cell response, anHLA-A30:01-restricted cytotoxic T cell response, anHLA-A31:01-restricted cytotoxic T cell response, anHLA-A33:01-restricted cytotoxic T cell response, anHLA-A33:03-restricted cytotoxic T cell response, anHLA-A68:01-restricted cytotoxic T cell response, or anHLA-A74:01-restricted cytotoxic T cell response; and that binds to anHLA-A02:01, HLA-A03:01, HLA-A11:01, HLA-A03:02, HLA-A30:01, HLA-A31:01,HLA-A33:01, HLA-A33:03, HLA-A68:01, and/or an HLA-A74:01; or at leastone polynucleotide encoding the at least one polypeptide.

In some aspects, provided herein is a composition comprising at leastone polypeptide comprising one or more mutant RAS peptide sequences eachcomprising: at least 8 contiguous amino acids of a mutant RAS proteincomprising a G12A, G12C, G12D, G12R, G12S, or G12V mutation, and theG12A, G12C, G12D, G12R, G12S, or G12V mutation; and further wherein thepeptide: comprises a mutation not encoded by a genome of a cancer celland has an affinity or predicted affinity of 150 nM or less for anHLA-A02:01 allele and/or a half-life of 2 hours or more, or has ahalf-life of 2 hours or more and an affinity or predicted affinity of150 nM or less for an HLA-A02:01 allele, an HLA-A03:01 allele, anHLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, anHLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, anHLA-A68:01 allele, or an HLA-A74:01 allele and/or an HLA-008:02 allele;or at least one polynucleotide encoding the at least one polypeptide.

In some embodiments, the composition further comprises (i) a peptidecomprising a peptide sequence in any one of Table 3 to 14, or (ii) apolynucleotide encoding the peptide comprising a sequence in Table 3 to14.

In some aspects, provided herein is a composition comprising: (a) atleast one polypeptide comprising one or more mutant RAS peptidesequences selected from the group consisting of: DTAGHEEY, TAGHEEYSAM,DILDTAGHE, DILDTAGH, ILDTAGHEE, ILDTAGHE, DILDTAGHEEY, DTAGHEEYS,LLDILDTAGH, DILDTAGRE, DILDTAGR, ILDTAGREE, ILDTAGRE, CLLDILDTAGR,TAGREEYSAM, REEYSAMRD, DTAGKEEYSAM, CLLDILDTAGK, DTAGKEEY, LLDILDTAGK,ILDTAGKE, ILDTAGKEE, DTAGLEEY, ILDTAGLE, DILDTAGL, ILDTAGLEE,GLEEYSAMRDQY, LLDILDTAGLE, LDILDTAGL, DILDTAGLE, DILDTAGLEEY, AGVGKSAL,GAAGVGKSAL, AAGVGKSAL, CGVGKSAL, ACGVGKSAL, DGVGKSAL, ADGVGKSAL,DGVGKSALTI, GARGVGKSA, KLVVVGARGV, VVVGARGV, SGVGKSAL, VVVGASGVGK,GASGVGKSAL, VGVGKSAL, VVVGAGCVGK, KLVVVGAGC, GDVGKSAL, DVGKSALTI,VVVGAGDVGK, TAGKEEYSAM, DTAGHEEYSAM, TAGHEEYSA, DTAGREEYSAM, TAGKEEYSA,AAGVGKSA, AGCVGKSAL, AGDVGKSAL, AGKEEYSAMR, AGVGKSALTI, ARGVGKSAL,ASGVGKSA, ASGVGKSAL, AVGVGKSA, CVGKSALTI, DILDTAGK, DILDTAGREEY,DTAGHEEYSAMR, DTAGKEEYS, DTAGKEEYSAMR, DTAGLEEYS, DTAGLEEYSA,DTAGLEEYSAMR, DTAGREEYS, DTAGREEYSAMR, GAAGVGKSA, GACGVGKSA, GACGVGKSAL,GADGVGKS, GAGDVGKSA, GAGDVGKSAL, GASGVGKSA, GCVGKSAL, GCVGKSALTI,GHEEYSAM, GKEEYSAM, GLEEYSAMR, GREEYSAM, GREEYSAMR, HEEYSAMRD,KEEYSAMRD, KLVVVGASG, LDILDTAGR, LEEYSAMRD, LVVVGARGV, LVVVGASGV,REEYSAMRDQY, RGVGKSAL, TAGLEEYSA, TEYKLVVVGAA, VGAAGVGKSA, VGADGVGK,VGASGVGKSA, VGVGKSALTI, VVVGAAGV, VVVGAVGV, YKLVVVGAC, YKLVVVGAD,YKLVVVGAR, and DILDTAGKE; or (b) at least one polynucleotide encodingthe at least one polypeptide.

In some embodiments, the composition further comprises (i) a peptidecomprising a peptide sequence in any one of Table 1 to 14, or (ii) apolynucleotide encoding the peptide comprising a sequence in Table 1 to14.

In some embodiments, at least one of the mutant RAS peptide sequencescomprises N or C terminal amino acid sequence extension of at least 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or40 amino acids, wherein the N or C terminal extension is a wild-type RASamino acid sequence or a non-heterologous RAS amino acid sequence.

In some embodiments, the at least one polypeptide comprises at least 3,4, 5, 6, 7, 8, 9, or 10 mutant RAS peptide sequences.

In some embodiments, the at least one polypeptide comprises at least twopolypeptides, or the at least one polynucleotide comprises at least twopolynucleotides.

In some embodiments, at least one of the mutant RAS peptide sequencescomprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,or 40 contiguous amino acids of a mutant RAS protein.

In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 of themutant RAS peptide sequences comprise at least 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, or 40 contiguous amino acids of a mutant RASprotein.

In some embodiments, each of the mutant RAS peptide sequences or each ofthe two or more RAS peptide sequences comprises at least 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous amino acids of amutant RAS protein.

In some embodiments, the at least one polypeptide comprises at least onemutant RAS peptide sequence that binds to or is predicted to bind to aprotein encoded by an HLA-A02:01 allele, an HLA-A03:01 allele, anHLA-A11:01 allele, and/or an HLA-008:02 allele.

In some embodiments, the at least one polypeptide comprises at least onemutant RAS peptide sequence that binds to or is predicted to bind to aprotein encoded by: an HLA-A02:01 allele and an HLA-A03:01 allele, anHLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, anHLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, anHLA-A68:01 allele, or an HLA-A74:01 allele; an HLA-A02:01 allele and anHLA-008:02 allele; an HLA-A03:01 allele, an HLA-A11:01 allele, anHLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, anHLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, or anHLA-A74:01 allele and an HLA-008:02 allele; or an HLA-A03:01 allele, anHLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, anHLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, anHLA-A68:01 allele, or an HLA-A74:01 allele and allele and an HLA-A03:01allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03allele, an HLA-A68:01 allele, or an HLA-A74:01 allele.

In some embodiments, the mutant RAS peptide sequences comprise a firstmutant RAS peptide sequence that binds to or is predicted to bind to aprotein encoded by an HLA-A02:01 allele, an HLA-A03:01 allele, anHLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, anHLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, anHLA-A68:01 allele, an HLA-A74:01 allele, and/or an HLA-008:02 allele;and a second RAS peptide sequence that binds to or is predicted to bindto a protein encoded by an HLA-A02:01 allele, an HLA-A03:01 allele, anHLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, anHLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, anHLA-A68:01 allele, an HLA-A74:01 allele, and/or an HLA-008:02 allele;wherein the first mutant RAS peptide sequence binds to or is predictedto bind to a protein encoded by different HLA allele than the secondmutant RAS peptide sequence.

In some embodiments, the at least one polypeptide comprises at least onemutant RAS peptide sequence that binds to a protein encoded by an HLAallele with an affinity of less than 10 μM, less than 1 μM, less than500 nM, less than 400 nM, less than 300 nM, less than 250 nM, less than200 nM, less than 150 nM, less than 100 nM, or less than 50 nM.

In some embodiments, the at least one polypeptide comprises at least onemutant RAS peptide sequence that binds to a protein encoded by an HLAallele with a stability of greater than 24 hours, greater than 12 hours,greater than 9 hours, greater than 6 hours, greater than 5 hours,greater than 4 hours, greater than 3 hours, greater than 2 hours,greater than 1 hour, greater than 45 minutes, greater than 30 minutes,greater than 15 minutes, or greater than 10 minutes.

In some embodiments, the HLA allele is selected from the groupconsisting of HLA-A02:01 allele, an HLA-A03:01 allele, an HLA-A11:01allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01allele, an HLA-A74:01 allele, and/or an HLA-008:02 allele and anycombination thereof.

In some embodiments, the at least one polypeptide comprises at least oneof the following sequences: LVVVGACGV, KLVVVGACGV, LVVVGADGV,KLVVVGADGV, LVVVGAVGV, KLVVVGAVGV, VVGACGVGK, VVVGACGVGK, VVGADGVGK,VVVGADGVGK, VVGAVGVGK, VVVGAVGVGK, VVGACGVGK, VVGADGVGK, VVVGADGVGK,VVGAVGVGK, and VVVGAVGVGK.

In some embodiments, the mutant RAS peptide sequences comprise at leastone or two of the following sequences: KLVVVGACGV, FLVVVGACGL,FMVVVGACGI, FLVVVGACGI, FMVVVGACGV, FLVVVGACGV, MLVVVGACGV, FMVVVGACGL,YLVVVGACGV, KMVVVGACGV, YMVVVGACGV, and MMVVVGACGV.

In some embodiments, the mutant RAS peptide sequences comprise at leastone or two of the following sequences: TEYKLVVVGAVGV;WQAGILARKLVVVGAVGVQGQNLKYQ; HSYTTAEKLVVVGAVGVILGVLLLI;PLTEEKIKKLVVVGAVGVEKEGKISK; GALHFKPGSRKLVVVGAVGVAASDFIFLVT;RRANKDATAEKLVVVGAVGVKELKQVASPF; KAFISHEEKRKLVVVGAVGVKKKLINEKKE;TDLSSRFSKSKLVVVGAVGVKKCDISLQFF; FDLGGGTFDVKLVVVGAVGVKSTAGDTHLG; orCLLLHYSVSKKLVVVGAVGVATFYVAVTVP.

In some embodiments, (Xaa)N comprises an amino acid sequence ofIDIIMKIRNA, FFFFFFFFFFFFFFFFFFFFIIFFIFFWMC,FFFFFFFFFFFFFFFFFFFFFFFFAAFWFW, IFFIFFIIFFFFFFFFFFFFIIIIIIIWEC,FIFFFIIFFFFFIFFFFFIFIIIIIIFWEC, TEY, WQAGILAR, HSYTTAE, PLTEEKIK,GALHFKPGSR, RRANKDATAE, KAFISHEEKR, TDLSSRFSKS, FDLGGGTFDV, CLLLHYSVSK,or MTEYKLVVV.

In some embodiments, (XaaC)C comprises an amino acid sequence ofKKNKKDDIKD, AGNDDDDDDDDDDDDDDDDDKKDKDDDDDD, AGNKKKKKKKNNNAGRDDDDDDDDDDDDDDDDDDDDDDDDDDD, GKSALTIQL, GKSALTI, QGQNLKYQ, ILGVLLLI,EKEGKISK, AASDFIFLVT, KELKQVASPF, KKKLINEKKE, KKCDISLQFF, KSTAGDTHLG,ATFYVAVTVP, LTIQLIQNHFVDEYDPTIEDSYRKQVVIDG, orTIQLIQNHFVDEYDPTIEDSYRKQVVIDGE.

In some embodiments, a first mutant RAS peptide sequence comprises afirst neoepitope of a mutant RAS protein and a second mutant RAS peptidesequence comprises a second neoepitope of a mutant RAS protein, whereinthe first mutant RAS peptide sequence is different from the mutant RASpeptide sequence, and wherein the first neoepitope comprises at leastone mutant amino acid and the second neoepitope comprises the samemutant amino acid.

In some embodiments, at least one of the mutant RAS peptide sequencescomprises a mutant amino acid not encoded by the genome of a cancer cellof a subject.

In some embodiments, each of the mutant RAS peptide sequences arepresent at a concentration at least 1 μg/mL, at least 10 μg/mL, at least25 μg/mL, at least 50 μg/mL, or at least 100 μg/mL.

In some embodiments, each of the mutant RAS peptide sequences arepresent at a concentration at most 5000 μg/mL, at most 2500 μg/mL, atmost 1000 μg/mL, at most 750 μg/mL, at most 500 μg/mL, at most 400μg/mL, or at most 300 μg/mL.

In some embodiments, each of the mutant RAS peptide sequences arepresent at a concentration of from 10 μg/mL to 5000 μg/mL, 10 μg/mL to4000 μg/mL, 10 μg/mL to 3000 μg/mL, 10 μg/mL to 2000 μg/mL, 10 μg/mL to1000 μg/mL, 25 μg/mL to 500 μg/mL, or 50 μg/mL to 300 μg/mL.

In some embodiments, the composition further comprises different mutantRAS peptide sequence with a G13A, G13C, G13D, G13R, G13S, G13V, G12A,G12C, G12D, G12R, G12S, G12V or a Q61 mutation.

In some embodiments, the composition further comprises animmunomodulatory agent or an adjuvant.

In some embodiments, the adjuvant is polyICLC.

In some aspects, provided herein is a pharmaceutical compositioncomprising: a composition described herein and a pharmaceuticallyacceptable excipient.

In some embodiments, the pharmaceutical composition comprises a pHmodifier present at a concentration of less than 1 mM or greater than 1mM.

In some embodiments, the pharmaceutical composition is a vaccinecomposition.

In some embodiments, the pharmaceutical composition is aqueous.

In some embodiments, one or more of the at least one polypeptide isbounded by pI>5 and HYDRO>−6, pI>8 and HYDRO >−8, pI<5 and HYDRO>−5,pI>9 and HYDRO<−8, pI>7 and a HYDRO value of >−5.5, pI<4.3 and−4≥HYDRO≥−8, pI>0 and HYDRO<−8, pI>0 and HYDRO>−4, or pI>4.3 and−4≥HYDRO≥−8, pI>0 and HYDRO>−4, or pI>4.3 and HYDRO≤−4., pI>0 andHYDRO>−4, or pI>4.3 and −4≥HYDRO≥−9, 5≥pI≥12 and −4≥HYDRO≥−9.

In some embodiments, the pH modifier is a base.

In some embodiments, the pH modifier is a conjugate base of a weak acid.

In some embodiments, the pH modifier is a pharmaceutically acceptablesalt.

In some embodiments, the pH modifier is a dicarboxylate ortricarboxylate salt.

In some embodiments, the pH modifier is citric acid and/or a citratesalt.

In some embodiments, the citrate salt is disodium citrate and/ortrisodium citrate.

In some embodiments, the pH modifier is succinic acid and/or a succinatesalt.

In some embodiments, the succinate salt is a disodium succinate and/or amonosodium succinate.

In some embodiments, the succinate salt is disodium succinatehexahydrate.

In some embodiments, the pH modifier is present at a concentration offrom 0.1 mM-1 mM.

In some embodiments, the pharmaceutically acceptable carrier comprises aliquid.

In some embodiments, the pharmaceutically acceptable carrier compriseswater.

In some embodiments, the pharmaceutically acceptable carrier comprises asugar.

In some embodiments, the sugar comprises dextrose or mannitol.

In some embodiments, the dextrose is present at a concentration of from1-10% w/v.

In some embodiments, the sugar comprises trehalose.

In some embodiments, the sugar comprises sucrose.

In some embodiments, the pharmaceutically acceptable carrier comprisesdimethyl sulfoxide (DMSO).

In some embodiments, the DMSO is present at a concentration from 0.1% to10%, 0.5% to 5%, or 1% to 3%.

In some embodiments, the pharmaceutically acceptable carrier does notcomprise dimethyl sulfoxide (DMSO).

In some embodiments, the pharmaceutical composition is lyophilizable.

In some embodiments, the pharmaceutical composition further comprises animmunomodulator or adjuvant.

In some embodiments, the immunomodulator or adjuvant is selected fromthe group consisting of poly-ICLC, 1018 ISS, aluminum salts, Amplivax,AS15, BCG, CP-870,893, CpG7909, CyaA, ARNAX, STING agonists, dSLIM,GM-CSF, FLT-3L, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS,ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryllipid A, MontanideIMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51,OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector system, PLGAmicroparticles, resiquimod, SRL172, Virosomes and other Virus-likeparticles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, and Aquila'sQS21 stimulon.

In some embodiments, the immunomodulator or adjuvant comprisespoly-ICLC.

In some embodiments, a ratio of poly-ICLC to peptides in thepharmaceutical composition is from 2:1 to 1:10 v:v.

In some embodiments, the ratio of poly-ICLC to peptides in thepharmaceutical composition is about 1:1, 1:2, 1:3, 1:4 or 1:5 v:v.

In some embodiments, the ratio of poly-ICLC to peptides in thepharmaceutical composition is about 1:3v:v.

In some aspects, provided herein is a method of treating a subject withcancer comprising administering to the subject a pharmaceuticalcomposition described herein.

In some aspects, provided herein is a method of treating a subject withcancer comprising administering to the subject a peptide with a sequenceof VVGADGVGK, VVGACGVGK, VVGAVGVGK, VVVGADGVGK, VVVGACGVGK, VVVGAVGVGK,wherein the subject expresses a protein encoded by an HLA-A02:01 allele,an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, anHLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, anHLA-A33:03 allele, an HLA-A68:01 allele, an HLA-A74:01 allele, or anHLA-008:02 allele of the subject's genome.

In some aspects, provided herein is a method of treating a subject withcancer comprising administering to the subject a mutant RAS peptide or anucleic acid encoding the mutant RAS peptide, wherein the mutant RASpeptide comprises at least 8 contiguous amino acids of a mutant RASprotein comprising a mutation at G12, wherein the peptide comprises themutation at G12 and binds to HLA-A11:01 or HLA-A03:01, wherein thesubject is identified as expressing a protein encoded by an HLA-A03:01allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03allele, an HLA-A68:01 allele, or an HLA-A74:01 allele.

In some aspects, provided herein is a method of treating a subject withcancer comprising administering to the subject a peptide comprising asequence GADGVGKSAL, GACGVGKSAL, GAVGVGKSAL, GADGVGKSA, GACGVGKSA, orGAVGVGKSA; wherein the subject expresses a protein encoded by anHLA-A02:01 allele, an HLA-A03:01 allele, an HLA-A11:01 allele, anHLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, anHLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, anHLA-A74:01 allele, or an HLA-008:02 allele of the subject's genome thatbinds to the peptide.

In some aspects, provided herein is a method of treating a subject withcancer comprising administering to the subject a first and a secondpeptide or a nucleic acid encoding the first and second peptide, whereinthe first and second peptides comprise at least two of: (1) KLVVVGADGV,KLVVVGACGV, KLVVVGAVGV, LVVVGADGV, LVVVGACGV, LVVVGAVGV; (2) GADGVGKSAL,GACGVGKSAL, GAVGVGKSAL, GADGVGKSA, GACGVGKSA, GAVGVGKSA; and (3)VVGADGVGK, VVGACGVGK, VVGAVGVGK, VVVGADGVGK, VVVGACGVGK, VVVGAVGVGK;wherein the subject's HLA allele expression is unknown at the time ofadministration.

In some aspects, provided herein is a method of treating a subject withcancer comprising administering to the subject a mutant RAS peptide or anucleic acid encoding the mutant RAS peptide, wherein the mutant RASpeptide comprises at least 8 contiguous amino acids of a mutant RASprotein comprising a G12C mutation, wherein the peptide comprises theG12C mutation, and further wherein the peptide comprises a stabilizingmutation not encoded by a genome of a cancer cell, wherein the subjectexpresses a protein encoded by an HLA-A02:01 allele, an HLA-A03:01allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03allele, an HLA-A68:01 allele, an HLA-A74:01 allele, or an HLA-008:02allele.

In some aspects, provided herein is a method of identifying a subjectwith cancer as a candidate for a therapeutic, the method comprisingidentifying the subject as a subject that expresses a protein encoded byan HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, anHLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, anHLA-A33:03 allele, an HLA-A68:01 allele, or an HLA-A74:01 allele,wherein the therapeutic is a mutant RAS peptide or a nucleic acidencoding the mutant RAS peptide, wherein the mutant RAS peptidecomprises at least 8 contiguous amino acids of a mutant RAS proteincomprising a mutation at G12, wherein the peptide comprises the mutationat G12 and binds to a protein encoded by an HLA-A03:01 allele, anHLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, anHLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, anHLA-A68:01 allele, or an HLA-A74:01 allele.

In some embodiments, the method further comprises administering thetherapeutic to the subject.

In some aspects, provided herein is a method of treating a subject withcancer, the method comprising: (a) identifying a first protein expressedby the subject, wherein the first protein is encoded by a first HLAallele of the subject and wherein the first HLA allele is an HLA alleleprovided in any one of Tables 1 to 14; and (b) administering to thesubject (i) a first mutant RAS peptide, wherein the first mutant RASpeptide is a peptide to the first HLA allele provided in any one ofTables 1 to 14, or (ii) a polynucleic acid encoding the first mutant RASpeptide.

In some embodiments, the method further comprises identifying a secondprotein expressed by the subject, wherein the second protein is encodedby a second HLA allele of the subject and wherein the second HLA alleleis an HLA allele provided in any one of Tables 1 to 14.

In some embodiments, the method further comprises administering to thesubject (i) a second mutant RAS peptide, wherein the second mutant RASpeptide is a peptide to the second HLA allele provided in any one ofTables 1 to 14, or (ii) a polynucleic acid encoding the second mutantRAS peptide.

In some embodiments, the first HLA allele is different from the secondHLA allele.

In some embodiments, the first mutant RAS peptide is different from thesecond mutant RAS peptide.

For example, in some embodiments, the first protein expressed by asubject is encoded by HLA-A03:01 as provided, e.g., in Table 11, and themethod comprises administering to the subject a first mutant RAS peptidecomprising a sequence of VVGASGVGK or a polynucleic acid encoding thefirst mutant RAS peptide.

For example, in some embodiments, the first protein expressed by asubject is encoded by HLA-A03:01 as provided, e.g., in Table 5, and themethod comprises administering to the subject a first mutant RAS peptidecomprising a sequence of CLLDILDTAGK or a polynucleic acid encoding thefirst mutant RAS peptide.

For another example, in some embodiments, the second protein expressedby a subject is encoded by HLA-A11:01 as provided, e.g., in Table 11,and the method comprises administering to the subject a second mutantRAS peptide comprising a sequence of VVVGASGVGK or a polynucleic acidencoding the second mutant RAS peptide.

For yet another example, in some embodiments, the second proteinexpressed by a subject is encoded by HLA-008:02 as provided, e.g., inTable 9, and the method comprises administering to the subject a secondmutant RAS peptide comprising a sequence of GADGVGKSAL or a polynucleicacid encoding the second mutant RAS peptide.

In some embodiments, an immune response is elicited in the subject.

In some embodiments, the immune response is a humoral response.

In some embodiments, the mutant RAS peptide sequences are administeredsimultaneously, separately or sequentially.

In some embodiments, the first peptide is sequentially administeredafter a time period sufficient for the second peptide to activate thesecond T cells.

In some embodiments, the cancer is selected from the group consisting oflung cancer, non-small cell lung cancer, pancreatic cancer, colorectalcancer, uterine cancer and liver cancer.

In some embodiments, the method further comprises comprisingadministering at least one additional therapeutic agent or modality.

In some embodiments, the at least one additional therapeutic agent ormodality is surgery, a checkpoint inhibitor, an antibody or fragmentthereof, a chemotherapeutic agent, radiation, a vaccine, a smallmolecule, a T cell, a vector, and APC, a polynucleotide, an oncolyticvirus or any combination thereof.

In some embodiments, the at least one additional therapeutic agent is ananti-PD-1 agent and anti-PD-L1 agent, an anti-CTLA-4 agent, or ananti-CD40 agent.

In some embodiments, the additional therapeutic agent is administeredbefore, simultaneously, or after administering the mutant RAS peptidesequences.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the present disclosure are set forth with particularityin the appended claims. A better understanding of the features andadvantages of the present will be obtained by reference to the followingdetailed description that sets forth illustrative embodiments, in whichthe principles of the disclosure are utilized, and the accompanyingdrawings of which:

FIG. 1 illustrates an exemplary workflow for determination of RASepitopes that can induce CD8+ and/or CD4+ T cells.

FIGS. 2A and 2B illustrate a summary of experiments showing thatpredicted RAS G12C epitopes to HLA-A11:01 (left), and RAS G12V epitopesto HLA-A11:01 (right) can be detected by mass spectrometry.

FIGS. 3A and 3B depict illustrative multimer plots of RAS-mutantspecific CD8+ T cell responses.

FIG. 3C depicts example results showing antigen specific CD8⁺ T cellresponses to a long peptide containing the minimal epitope for KRAS G12Von HLA-A11:01. The sequences of the long peptide used to stimulate theresponse is indicated, as well as the sequence of the minimal epitopeused for the multimer staining.

FIG. 4A is a graph showing antigen-specific induction of IFNγ. The IFNγlevels of samples mock transduced or transduced with a lentiviralexpression vector encoding a mutant RAS peptide are shown.

FIG. 4B is a graph showing upregulation of active caspase 3 on targetcells. The percent live caspase-A positive target cells of samples mocktransduced or transduced with a lentiviral expression vector encoding amutant RAS peptide are shown.

FIG. 5A is a graph showing antigen-specific induction of IL-2 byco-culturing T cells expressing a TCR specific to a mutant RAS peptidewith 9mer or 11mer mutant RAS peptide-transduced target cells. The datashows that RAS specific T cells recognize mutated cells and upregulatecytotoxic machinery.

FIG. 5B is a graph showing antigen-specific induction of IL-2 byco-culturing T cells expressing a TCR specific to a 9mer or 11mer mutantRAS peptide with target cells loaded with increasing concentrations of amutant RAS peptide. The data shows that RAS specific T cells recognizemutated cells and upregulate cytotoxic machinery.

FIG. 5C is a graph showing antigen-specific induction of IL-2 byco-culturing T cells expressing a TCR specific to a mutant RAS peptidewith 9mer mutant RAS peptide-transduced target cells. The data showsthat RAS specific T cells recognize mutated cells and upregulatecytotoxic machinery.

FIG. 5D is a graph showing antigen-specific induction of IL-2 byco-culturing T cells expressing a TCR specific to a 9mer mutant RASpeptide with target cells loaded with increasing concentrations of amutant RAS peptide. The data shows that RAS specific T cells recognizemutated cells and upregulate cytotoxic machinery.

FIGS. 5E-5H demonstrate antigen-specific cytotoxicity activity of Tcells expressing a TCR specific to mutant RAS peptides. The data showsthat RAS specific TCRs can elicit specific recognition of cells carryingthe mutated peptide and the proper MHC-I and upregulate cytotoxicmachinery.

FIG. 6 depicts a FACS analysis of antigen-specific induction of IFNγlevels of CD4+ cells from a healthy donor stimulated with APCs loadedwith or without a mutant RAS peptide.

DETAILED DESCRIPTION

Described herein are new immunotherapeutic agents and uses thereof basedon the discovery of neoantigens arising from mutational events unique toan individual's tumor. Accordingly, the present disclosure describedherein provides peptides, polynucleotides encoding the peptides, andpeptide binding agents that can be used, for example, to stimulate animmune response to a tumor associated antigen or neoepitope, to createan immunogenic composition or cancer vaccine for use in treatingdisease.

The following description and examples illustrate embodiments of thepresent disclosure in detail. It is to be understood that this presentdisclosure is not limited to the particular embodiments described hereinand as such can vary. Those of skill in the art will recognize thatthere are numerous variations and modifications of this presentdisclosure, which are encompassed within its scope.

All terms are intended to be understood as they would be understood by aperson skilled in the art. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which the disclosurepertains.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Although various features of the present disclosure may be described inthe context of a single embodiment, the features may also be providedseparately or in any suitable combination. Conversely, although thepresent disclosure may be described herein in the context of separateembodiments for clarity, the present disclosure may also be implementedin a single embodiment.

The following definitions supplement those in the art and are directedto the current application and are not to be imputed to any related orunrelated case, e.g., to any commonly owned patent or application.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice for testing of the presentdisclosure, the preferred materials and methods are described herein.Accordingly, the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

I. Definitions

The terminology used herein is for the purpose of describing particularcases only and is not intended to be limiting. In this application, theuse of the singular includes the plural unless specifically statedotherwise. As used herein, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise.

In this application, the use of “or” means “and/or” unless statedotherwise. The terms “and/or” and “any combination thereof” and theirgrammatical equivalents as used herein, can be used interchangeably.These terms can convey that any combination is specificallycontemplated. Solely for illustrative purposes, the following phrases“A, B, and/or C” or “A, B, C, or any combination thereof” can mean “Aindividually; B individually; C individually; A and B; B and C; A and C;and A, B, and C.” The term “or” can be used conjunctively ordisjunctively, unless the context specifically refers to a disjunctiveuse.

The term “about” or “approximately” can mean within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, up to 10%, up to 5%, or up to 1% of a given value.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, within5-fold, and more preferably within 2-fold, of a value. Where particularvalues are described in the application and claims, unless otherwisestated the term “about” meaning within an acceptable error range for theparticular value should be assumed.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps. It is contemplated that any embodimentdiscussed in this specification can be implemented with respect to anymethod or composition of the present disclosure, and vice versa.Furthermore, compositions of the present disclosure can be used toachieve methods of the present disclosure.

Reference in the specification to “some embodiments,” “an embodiment,”“one embodiment” or “other embodiments” means that a particular feature,structure, or characteristic described in connection with theembodiments is included in at least some embodiments, but notnecessarily all embodiments, of the present disclosures. To facilitatean understanding of the present disclosure, a number of terms andphrases are defined below.

“Major Histocompatibility Complex” or “MHC” is a cluster of genes thatplays a role in control of the cellular interactions responsible forphysiologic immune responses. In humans, the MHC complex is also knownas the human leukocyte antigen (HLA) complex. For a detailed descriptionof the MHC and HLA complexes, see, Paul, Fundamental Immunology, 3^(rd)Ed., Raven Press, New York (1993). “Proteins or molecules of the majorhistocompatibility complex (MHC)”, “MHC molecules”, “MHC proteins” or“HLA proteins” are to be understood as meaning proteins capable ofbinding peptides resulting from the proteolytic cleavage of proteinantigens and representing potential lymphocyte epitopes, (e.g., T cellepitope and B cell epitope) transporting them to the cell surface andpresenting them there to specific cells, in particular cytotoxicT-lymphocytes, T-helper cells, or B cells. The major histocompatibilitycomplex in the genome comprises the genetic region whose gene productsexpressed on the cell surface are important for binding and presentingendogenous and/or foreign antigens and thus for regulating immunologicalprocesses. The major histocompatibility complex is classified into twogene groups coding for different proteins, namely molecules of MHC classI and molecules of MHC class II. The cellular biology and the expressionpatterns of the two MHC classes are adapted to these different roles.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II MajorHistocompatibility Complex (MHC) protein (see, e.g., Stites, et al.,Immunology, 8th Ed., Lange Publishing, Los Altos, Calif. (1994).

“Polypeptide”, “peptide” and their grammatical equivalents as usedherein refer to a polymer of amino acid residues, typically L-aminoacids, connected one to the other, typically by peptide bonds betweenthe α-amino and carboxyl groups of adjacent amino acids. Polypeptidesand peptides include, but are not limited to, mutant peptides,“neoantigen peptides” and “neoantigenic peptides”. Polypeptides orpeptides can be a variety of lengths, either in their neutral(uncharged) forms or in forms which are salts, and either free ofmodifications such as glycosylation, side chain oxidation, orphosphorylation or containing these modifications, subject to thecondition that the modification not destroy the biological activity ofthe polypeptides as herein described. A “mature protein” is a proteinwhich is full-length and which, optionally, includes glycosylation orother modifications typical for the protein in a given cellularenvironment. Polypeptides and proteins disclosed herein (includingfunctional portions and functional variants thereof) can comprisesynthetic amino acids in place of one or more naturally-occurring aminoacids. Such synthetic amino acids are known in the art, and include, forexample, aminocyclohexane carboxylic acid, norleucine, α-aminon-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- andtrans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine,4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserineβ-hydroxyphenylalanine, phenylglycine, α-naphthylalanine,cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid,aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine,N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid,α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine,and α-tert-butylglycine. The present disclosure further contemplatesthat expression of polypeptides described herein in an engineered cellcan be associated with post-translational modifications of one or moreamino acids of the polypeptide constructs. Non-limiting examples ofpost-translational modifications include phosphorylation, acylationincluding acetylation and formylation, glycosylation (including N-linkedand O-linked), amidation, hydroxylation, alkylation includingmethylation and ethylation, ubiquitination, addition of pyrrolidonecarboxylic acid, formation of disulfide bridges, sulfation,myristoylation, palmitoylation, isoprenylation, farnesylation,geranylation, glypiation, lipoylation and iodination.

A peptide or polypeptide may comprise at least one flanking sequence.The term “flanking sequence” as used herein refers to a fragment orregion of a peptide that is not a part of an epitope.

An “immunogenic” peptide or an “immunogenic” epitope or “peptideepitope” is a peptide that comprises an allele-specific motif such thatthe peptide will bind an HLA molecule and induce a cell-mediated orhumoral response, for example, cytotoxic T lymphocyte (CTL (e.g.,CD8⁺)), helper T lymphocyte (Th (e.g., CD4⁺)) and/or B lymphocyteresponse. Thus, immunogenic peptides described herein are capable ofbinding to an appropriate HLA molecule and thereafter inducing a CTL(cytotoxic) response, or a HTL (and humoral) response, to the peptide.

“Neoantigen” means a class of tumor antigens which arise fromtumor-specific changes in proteins. Neoantigens encompass, but are notlimited to, tumor antigens which arise from, for example, substitutionin the protein sequence, frame shift mutation, fusion polypeptide,in-frame deletion, insertion, expression of endogenous retroviralpolypeptides, and tumor-specific overexpression of polypeptides.

The term “residue” refers to an amino acid residue or amino acid mimeticresidue incorporated into a peptide or protein by an amide bond or amidebond mimetic, or nucleic acid (DNA or RNA) that encodes the amino acidor amino acid mimetic.

A “neoepitope”, “tumor specific neoepitope” or “tumor antigen” refers toan epitope or antigenic determinant region that is not present in areference, such as a non-diseased cell, e.g., a non-cancerous cell or agermline cell, but is found in a diseased cell, e.g., a cancer cell.This includes situations where a corresponding epitope is found in anormal non-diseased cell or a germline cell but, due to one or moremutations in a diseased cell, e.g., a cancer cell, the sequence of theepitope is changed so as to result in the neoepitope. The term“neoepitope” as used herein refers to an antigenic determinant regionwithin the peptide or neoantigenic peptide. A neoepitope may comprise atleast one “anchor residue” and at least one “anchor residue flankingregion.” A neoepitope may further comprise a “separation region.” Theterm “anchor residue” refers to an amino acid residue that binds tospecific pockets on HLAs, resulting in specificity of interactions withHLAs. In some cases, an anchor residue may be at a canonical anchorposition. In other cases, an anchor residue may be at a non-canonicalanchor position. Neoepitopes may bind to HLA molecules through primaryand secondary anchor residues protruding into the pockets in thepeptide-binding grooves. In the peptide-binding grooves, specific aminoacids compose pockets that accommodate the corresponding side chains ofthe anchor residues of the presented neoepitopes. Peptide-bindingpreferences exist among different alleles of both of HLA I and HLA IImolecules. HLA class I molecules bind short neoepitopes, whose N- andC-terminal ends are anchored into the pockets located at the ends of theneoepitope binding groove. While the majority of the HLA class I bindingneoepitopes are of about 9 amino acids, longer neoepitopes can beaccommodated by the bulging of their central portion, resulting inbinding neoepitopes of about 8 to 12 amino acids. Neoepitopes binding toHLA class II proteins are not constrained in size and can vary fromabout 16 to 25 amino acids. The neoepitope binding groove in the HLAclass II molecules is open at both ends, which enables binding ofpeptides with relatively longer length. Though the core 9 amino acidresidues long segment contributes the most to the recognition of theneoepitope, the anchor residue flanking regions are also important forthe specificity of the peptide to the HLA class II allele. In somecases, the anchor residue flanking region is N-terminus residues. Inanother case, the anchor residue flanking region is C-terminus residues.In yet another case, the anchor residue flanking region is bothN-terminus residues and C-terminus residues. In some cases, the anchorresidue flanking region is flanked by at least two anchor residues. Ananchor residue flanking region flanked by anchor residues is a“separation region.”

A “reference” can be used to correlate and compare the results obtainedin the methods of the present disclosure from a tumor specimen.Typically the “reference” may be obtained on the basis of one or morenormal specimens, in particular specimens which are not affected by acancer disease, either obtained from a patient or one or more differentindividuals, for example, healthy individuals, in particular individualsof the same species. A “reference” can be determined empirically bytesting a sufficiently large number of normal specimens.

An “epitope” is the collective features of a molecule, such as primary,secondary and tertiary peptide structure, and charge, that together forma site recognized by, for example, an immunoglobulin, T cell receptor,HLA molecule, or chimeric antigen receptor. Alternatively, an epitopecan be defined as a set of amino acid residues which is involved inrecognition by a particular immunoglobulin, or in the context of Tcells, those residues necessary for recognition by T cell receptorproteins, chimeric antigen receptors, and/or Major HistocompatibilityComplex (MHC) receptors. A “T cell epitope” is to be understood asmeaning a peptide sequence which can be bound by the MHC molecules ofclass I or II in the form of a peptide-presenting MHC molecule or MHCcomplex and then, in this form, be recognized and bound by T cells, suchas T-lymphocytes or T-helper cells. Epitopes can be prepared byisolation from a natural source, or they can be synthesized according tostandard protocols in the art. Synthetic epitopes can compriseartificial amino acid residues, “amino acid mimetics,” such as D isomersof naturally-occurring L amino acid residues or non-naturally-occurringamino acid residues such as cyclohexylalanine. Throughout thisdisclosure, epitopes may be referred to in some cases as peptides orpeptide epitopes. It is to be appreciated that proteins or peptides thatcomprise an epitope or an analog described herein as well as additionalamino acid(s) are still within the bounds of the present disclosure. Incertain embodiments, the peptide comprises a fragment of an antigen. Incertain embodiments, there is a limitation on the length of a peptide ofthe present disclosure. The embodiment that is length-limited occurswhen the protein or peptide comprising an epitope described hereincomprises a region (i.e., a contiguous series of amino acid residues)having 100% identity with a native sequence. In order to avoid thedefinition of epitope from reading, e.g., on whole natural molecules,there is a limitation on the length of any region that has 100% identitywith a native peptide sequence. Thus, for a peptide comprising anepitope described herein and a region with 100% identity with a nativepeptide sequence, the region with 100% identity to a native sequencegenerally has a length of: less than or equal to 600 amino acidresidues, less than or equal to 500 amino acid residues, less than orequal to 400 amino acid residues, less than or equal to 250 amino acidresidues, less than or equal to 100 amino acid residues, less than orequal to 85 amino acid residues, less than or equal to 75 amino acidresidues, less than or equal to 65 amino acid residues, and less than orequal to 50 amino acid residues. In certain embodiments, an “epitope”described herein is comprised by a peptide having a region with lessthan 51 amino acid residues that has 100% identity to a native peptidesequence, in any increment down to 5 amino acid residues; for example50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33,32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues.

The nomenclature used to describe peptides or proteins follows theconventional practice wherein the amino group is presented to the left(the amino- or N-terminus) and the carboxyl group to the right (thecarboxy- or C-terminus) of each amino acid residue. When amino acidresidue positions are referred to in a peptide epitope they are numberedin an amino to carboxyl direction with position one being the residuelocated at the amino terminal end of the epitope, or the peptide orprotein of which it can be a part. In the formula representing selectedspecific embodiments of the present disclosure, the amino- andcarboxyl-terminal groups, although not specifically shown, are in theform they would assume at physiologic pH values, unless otherwisespecified. In the amino acid structure formula, each residue isgenerally represented by standard three letter or single letterdesignations. The L-form of an amino acid residue is represented by acapital single letter or a capital first letter of a three-lettersymbol, and the D-form for those amino acid residues having D-forms isrepresented by a lower case single letter or a lower case three lettersymbol. However, when three letter symbols or full names are usedwithout capitals, they can refer to L amino acid residues. Glycine hasno asymmetric carbon atom and is simply referred to as “Gly” or “G”. Theamino acid sequences of peptides set forth herein are generallydesignated using the standard single letter symbol. (A, Alanine; C,Cysteine; D, Aspartic Acid; E, Glutamic Acid; F, Phenylalanine; G,Glycine; H, Histidine; I, Isoleucine; K, Lysine; L, Leucine; M,Methionine; N, Asparagine; P, Proline; Q, Glutamine; R, Arginine; S,Serine; T, Threonine; V, Valine; W, Tryptophan; and Y, Tyrosine.)

The term “mutation” refers to a change of or difference in the nucleicacid sequence (nucleotide substitution, addition or deletion) comparedto a reference. A “somatic mutation” can occur in any of the cells ofthe body except the germ cells (sperm and egg) and therefore are notpassed on to children. These alterations can (but do not always) causecancer or other diseases. In some embodiments, a mutation is anon-synonymous mutation. The term “non-synonymous mutation” refers to amutation, for example, a nucleotide substitution, which does result inan amino acid change such as an amino acid substitution in thetranslation product. A “frameshift” occurs when a mutation disrupts thenormal phase of a gene's codon periodicity (also known as “readingframe”), resulting in the translation of a non-native protein sequence.It is possible for different mutations in a gene to achieve the samealtered reading frame.

A “conservative” amino acid substitution is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. Methods ofidentifying nucleotide and amino acid conservative substitutions whichdo not eliminate peptide function are well-known in the art.

As used herein, the term “affinity” refers to a measure of the strengthof binding between two members of a binding pair, for example, anHLA-binding peptide and a class I or II HLA. K_(D) is the dissociationconstant and has units of molarity. The affinity constant is the inverseof the dissociation constant. An affinity constant is sometimes used asa generic term to describe this chemical entity. It is a direct measureof the energy of binding Affinity may be determined experimentally, forexample by surface plasmon resonance (SPR) using commercially availableBiacore SPR units. Affinity may also be expressed as the inhibitoryconcentration 50 (IC₅₀), that concentration at which 50% of the peptideis displaced. Likewise, ln(IC₅₀) refers to the natural log of the IC₅₀.K_(off) refers to the off-rate constant, for example, for dissociationof an HLA-binding peptide and a class I or II HLA. Throughout thisdisclosure, “binding data” results can be expressed in terms of “IC₅₀.”IC₅₀ is the concentration of the tested peptide in a binding assay atwhich 50% inhibition of binding of a labeled reference peptide isobserved. Given the conditions in which the assays are run (i.e.,limiting HLA protein and labeled reference peptide concentrations),these values approximate K_(D) values. Assays for determining bindingare well known in the art and are described in detail, for example, inPCT publications WO 94/20127 and WO 94/03205, and other publicationssuch Sidney et al., Current Protocols in Immunology 18.3.1 (1998);Sidney, et al., J. Immunol. 154:247 (1995); and Sette, et al., Mol.Immunol. 31:813 (1994). Alternatively, binding can be expressed relativeto binding by a reference standard peptide. For example, can be based onits IC₅₀, relative to the IC₅₀ of a reference standard peptide. Bindingcan also be determined using other assay systems including those using:live cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnicket al., Nature 352:67 (1991); Busch et al., Int. Immunol. 2:443 (1990);Hill et al., J. Immunol. 147:189 (1991); del Guercio et al., J. Immunol.154:685 (1995)), cell free systems using detergent lysates (e.g.,Cerundolo et al., J. Immunol. 21:2069 (1991)), immobilized purified MHC(e.g., Hill et al., J. Immunol. 152, 2890 (1994); Marshall et al., J.Immunol. 152:4946 (1994)), ELISA systems (e.g., Reay et al., EMBO J.11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J.Biol. Chem. 268:15425 (1993)); high flux soluble phase assays (Hammer etal., J. Exp. Med. 180:2353 (1994)), and measurement of class I MHCstabilization or assembly (e.g., Ljunggren et al., Nature 346:476(1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell62:285 (1990); Parker et al., J. Immunol. 149:1896 (1992)).“Cross-reactive binding” indicates that a peptide is bound by more thanone HLA molecule; a synonym is degenerate binding.

The term “derived” and its grammatical equivalents when used to discussan epitope is a synonym for “prepared” and its grammatical equivalents.A derived epitope can be isolated from a natural source, or it can besynthesized according to standard protocols in the art. Syntheticepitopes can comprise artificial amino acid residues “amino acidmimetics,” such as D isomers of natural occurring L amino acid residuesor non-natural amino acid residues such as cyclohexylalanine A derivedor prepared epitope can be an analog of a native epitope.

A “native” or a “wild type” sequence refers to a sequence found innature. Such a sequence can comprise a longer sequence in nature.

A “receptor” is to be understood as meaning a biological molecule or amolecule grouping capable of binding a ligand. A receptor may serve, totransmit information in a cell, a cell formation or an organism. Thereceptor comprises at least one receptor unit, for example, where eachreceptor unit may consist of a protein molecule. The receptor has astructure which complements that of a ligand and may complex the ligandas a binding partner. The information is transmitted in particular byconformational changes of the receptor following complexation of theligand on the surface of a cell. In some embodiments, a receptor is tobe understood as meaning in particular proteins of MHC classes I and IIcapable of forming a receptor/ligand complex with a ligand, inparticular a peptide or peptide fragment of suitable length.

A “ligand” is to be understood as meaning a molecule which has astructure complementary to that of a receptor and is capable of forminga complex with this receptor. In some embodiments, a ligand is to beunderstood as meaning a peptide or peptide fragment which has a suitablelength and suitable binding motifs in its amino acid sequence, so thatthe peptide or peptide fragment is capable of forming a complex withproteins of MHC class I or MHC class II.

In some embodiments, a “receptor/ligand complex” is also to beunderstood as meaning a “receptor/peptide complex” or “receptor/peptidefragment complex”, including a peptide- or peptide fragment-presentingMHC molecule of class I or of class II.

“Synthetic peptide” refers to a peptide that is obtained from anon-natural source, e.g., is man-made. Such peptides can be producedusing such methods as chemical synthesis or recombinant DNA technology.“Synthetic peptides” include “fusion proteins.”

The term “motif” refers to a pattern of residues in an amino acidsequence of defined length, for example, a peptide of less than about 15amino acid residues in length, or less than about 13 amino acid residuesin length, for example, from about 8 to about 13 amino acid residues(e.g., 8, 9, 10, 11, 12, or 13) for a class I HLA motif and from about 6to about 25 amino acid residues (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) for a class II HLA motif,which is recognized by a particular HLA molecule. Motifs are typicallydifferent for each HLA protein encoded by a given human HLA allele.These motifs differ in their pattern of the primary and secondary anchorresidues. In some embodiments, an MHC class I motif identifies a peptideof 9, 10, or 11 amino acid residues in length.

The term “naturally occurring” and its grammatical equivalents as usedherein refer to the fact that an object can be found in nature. Forexample, a peptide or nucleic acid that is present in an organism(including viruses) and can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally occurring.

According to the present disclosure, the term “vaccine” relates to apharmaceutical preparation (pharmaceutical composition) or product thatupon administration induces an immune response, for example, a cellularor humoral immune response, which recognizes and attacks a pathogen or adiseased cell such as a cancer cell. A vaccine may be used for theprevention or treatment of a disease. The term “individualized cancervaccine” or “personalized cancer vaccine” concerns a particular cancerpatient and means that a cancer vaccine is adapted to the needs orspecial circumstances of an individual cancer patient.

A “protective immune response” or “therapeutic immune response” refersto a CTL and/or an HTL response to an antigen derived from an pathogenicantigen (e.g., a tumor antigen), which in some way prevents or at leastpartially arrests disease symptoms, side effects or progression. Theimmune response can also include an antibody response which has beenfacilitated by the stimulation of helper T cells.

“Antigen processing” or “processing” and its grammatical equivalentsrefers to the degradation of a polypeptide or antigen into processionproducts, which are fragments of said polypeptide or antigen (e.g., thedegradation of a polypeptide into peptides) and the association of oneor more of these fragments (e.g., via binding) with MHC molecules forpresentation by cells, for example, antigen presenting cells, tospecific T cells.

“Antigen presenting cells” (APC) are cells which present peptidefragments of protein antigens in association with MHC molecules on theircell surface. Some APCs may activate antigen specific T cells.Professional antigen-presenting cells are very efficient atinternalizing antigen, either by phagocytosis or by receptor-mediatedendocytosis, and then displaying a fragment of the antigen, bound to aclass II MHC molecule, on their membrane. The T cell recognizes andinteracts with the antigen-class II MHC molecule complex on the membraneof the antigen presenting cell. An additional co-stimulatory signal isthen produced by the antigen presenting cell, leading to activation ofthe T cell. The expression of co-stimulatory molecules is a definingfeature of professional antigen-presenting cells. The main types ofprofessional antigen-presenting cells are dendritic cells, which havethe broadest range of antigen presentation, and are probably the mostimportant antigen presenting cells, macrophages, B-cells, and certainactivated epithelial cells. Dendritic cells (DCs) are leukocytepopulations that present antigens captured in peripheral tissues to Tcells via both MHC class II and I antigen presentation pathways. It iswell known that dendritic cells are potent inducers of immune responsesand the activation of these cells is a critical step for the inductionof antitumoral immunity. Dendritic cells are conveniently categorized as“immature” and “mature” cells, which can be used as a simple way todiscriminate between two well characterized phenotypes. However, thisnomenclature should not be construed to exclude all possibleintermediate stages of differentiation. Immature dendritic cells arecharacterized as antigen presenting cells with a high capacity forantigen uptake and processing, which correlates with the high expressionof Fc receptor (FcR) and mannose receptor. The mature phenotype istypically characterized by a lower expression of these markers, but ahigh expression of cell surface molecules responsible for T cellactivation such as class I and class II MHC, adhesion molecules (e.g.,CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and4-1 BB).

The terms “identical” and its grammatical equivalents as used herein or“sequence identity” in the context of two nucleic acid sequences oramino acid sequences of polypeptides refers to the residues in the twosequences which are the same when aligned for maximum correspondenceover a specified comparison window. A “comparison window”, as usedherein, refers to a segment of at least about 20 contiguous positions,usually about 50 to about 200, more usually about 100 to about 150 inwhich a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are alignedoptimally. Methods of alignment of sequences for comparison arewell-known in the art. Optimal alignment of sequences for comparison maybe conducted by the local homology algorithm of Smith and Waterman, Adv.Appl. Math., 2:482 (1981); by the alignment algorithm of Needleman andWunsch, J. Mol. Biol., 48:443 (1970); by the search for similaritymethod of Pearson and Lipman, Proc. Nat. Acad. Sci. U.S.A., 85:2444(1988); by computerized implementations of these algorithms (including,but not limited to CLUSTAL in the PC/Gene program by Intelligentics,Mountain View Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in theWisconsin Genetics Software Package, Genetics Computer Group (GCG), 575Science Dr., Madison, Wis., U.S.A.); the CLUSTAL program is welldescribed by Higgins and Sharp, Gene, 73:237-244 (1988) and Higgins andSharp, CABIOS, 5:151-153 (1989); Corpet et al., Nucleic Acids Res.,16:10881-10890 (1988); Huang et al., Computer Applications in theBiosciences, 8:155-165 (1992); and Pearson et al., Methods in MolecularBiology, 24:307-331 (1994). Alignment is also often performed byinspection and manual alignment. In one class of embodiments, thepolypeptides herein have at least 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% sequence identity to a referencepolypeptide, or a fragment thereof, e.g., as measured by BLASTP (orCLUSTAL, or any other available alignment software) using defaultparameters. Similarly, nucleic acids can also be described withreference to a starting nucleic acid, e.g., they can have 50%, 60%, 70%,75%, 80%, 85%, 90%, 98%, 99% or 100% sequence identity to a referencenucleic acid or a fragment thereof, e.g., as measured by BLASTN (orCLUSTAL, or any other available alignment software) using defaultparameters. When one molecule is said to have certain percentage ofsequence identity with a larger molecule, it means that when the twomolecules are optimally aligned, said percentage of residues in thesmaller molecule finds a match residue in the larger molecule inaccordance with the order by which the two molecules are optimallyaligned.

The term “substantially identical” and its grammatical equivalents asapplied to nucleic acid or amino acid sequences mean that a nucleic acidor amino acid sequence comprises a sequence that has at least 90%sequence identity or more, at least 95%, at least 98% and at least 99%,compared to a reference sequence using the programs described above,e.g., BLAST, using standard parameters. For example, the BLASTN program(for nucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word length(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)).Percentage of sequence identity is determined by comparing two optimallyaligned sequences over a comparison window, wherein the portion of thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) as compared to the reference sequence (whichdoes not comprise additions or deletions) for optimal alignment of thetwo sequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. In embodiments, thesubstantial identity exists over a region of the sequences that is atleast about 50 residues in length, over a region of at least about 100residues, and in embodiments, the sequences are substantially identicalover at least about 150 residues. In embodiments, the sequences aresubstantially identical over the entire length of the coding regions.

The term “vector” as used herein means a construct, which is capable ofdelivering, and usually expressing, one or more gene(s) or sequence(s)of interest in a host cell. Examples of vectors include, but are notlimited to, viral vectors, naked DNA or RNA expression vectors, plasmid,cosmid, or phage vectors, DNA or RNA expression vectors associated withcationic condensing agents, and DNA or RNA expression vectorsencapsulated in liposomes.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionwhich is “isolated” is a polypeptide, antibody, polynucleotide, vector,cell, or composition which is in a form not found in nature. Isolatedpolypeptides, antibodies, polynucleotides, vectors, cells, orcompositions include those which have been purified to a degree thatthey are no longer in a form in which they are found in nature. In someembodiments, a polypeptide, antibody, polynucleotide, vector, cell, orcomposition which is isolated is substantially pure. In someembodiments, an “isolated polynucleotide” encompasses a PCR orquantitative PCR reaction comprising the polynucleotide amplified in thePCR or quantitative PCR reaction.

The term “isolated”, “biologically pure” or their grammaticalequivalents refers to material which is substantially or essentiallyfree from components which normally accompany the material as it isfound in its native state. Thus, isolated peptides described herein donot contain some or all of the materials normally associated with thepeptides in their in situ environment. An “isolated” epitope refers toan epitope that does not include the whole sequence of the antigen fromwhich the epitope was derived. Typically the “isolated” epitope does nothave attached thereto additional amino acid residues that result in asequence that has 100% identity over the entire length of a nativesequence. The native sequence can be a sequence such as atumor-associated antigen from which the epitope is derived. Thus, theterm “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). An “isolated” nucleic acid is a nucleic acid removed fromits natural environment. For example, a naturally-occurringpolynucleotide or peptide present in a living animal is not isolated,but the same polynucleotide or peptide, separated from some or all ofthe coexisting materials in the natural system, is isolated. Such apolynucleotide could be part of a vector, and/or such a polynucleotideor peptide could be part of a composition, and still be “isolated” inthat such vector or composition is not part of its natural environment.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules described herein, and further include such moleculesproduced synthetically.

The term “substantially purified” and its grammatical equivalents asused herein refer to a nucleic acid sequence, polypeptide, protein orother compound which is essentially free, i.e., is more than about 50%free of, more than about 70% free of, more than about 90% free of, thepolynucleotides, proteins, polypeptides and other molecules that thenucleic acid, polypeptide, protein or other compound is naturallyassociated with.

The term “substantially pure” as used herein refers to material which isat least 50% pure (i.e., free from contaminants), at least 90% pure, atleast 95% pure, at least 98% pure, or at least 99% pure.

The terms “polynucleotide”, “nucleotide”, “nucleic acid”, “polynucleicacid” or “oligonucleotide” and their grammatical equivalents are usedinterchangeably herein and refer to polymers of nucleotides of anylength, and include DNA and RNA, for example, mRNA. Thus, these termsincludes double and single stranded DNA, triplex DNA, as well as doubleand single stranded RNA. It also includes modified, for example, bymethylation and/or by capping, and unmodified forms of thepolynucleotide. The term is also meant to include molecules that includenon-naturally occurring or synthetic nucleotides as well as nucleotideanalogs. The nucleic acid sequences and vectors disclosed orcontemplated herein may be introduced into a cell by, for example,transfection, transformation, or transduction. The nucleotides can bedeoxyribonucleotides, ribonucleotides, modified nucleotides or bases,and/or their analogs, or any substrate that can be incorporated into apolymer by DNA or RNA polymerase. In some embodiments, thepolynucleotide and nucleic acid can be in vitro transcribed mRNA. Insome embodiments, the polynucleotide that is administered using themethods of the present disclosure is mRNA.

“Transfection,” “transformation,” or “transduction” as used herein referto the introduction of one or more exogenous polynucleotides into a hostcell by using physical or chemical methods. Many transfection techniquesare known in the art and include, for example, calcium phosphate DNAco-precipitation (see, e.g., Murray E. J. (ed.), Methods in MolecularBiology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press(1991)); DEAE-dextran; electroporation; cationic liposome-mediatedtransfection; tungsten particle-facilitated microparticle bombardment(Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNAco-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).Phage or viral vectors can be introduced into host cells, after growthof infectious particles in suitable packaging cells, many of which arecommercially available.

Nucleic acids and/or nucleic acid sequences are “homologous” when theyare derived, naturally or artificially, from a common ancestral nucleicacid or nucleic acid sequence. Proteins and/or protein sequences are“homologous” when their encoding DNAs are derived, naturally orartificially, from a common ancestral nucleic acid or nucleic acidsequence. The homologous molecules can be termed homologs. For example,any naturally occurring proteins, as described herein, can be modifiedby any available mutagenesis method. When expressed, this mutagenizednucleic acid encodes a polypeptide that is homologous to the proteinencoded by the original nucleic acid. Homology is generally inferredfrom sequence identity between two or more nucleic acids or proteins (orsequences thereof). The precise percentage of identity between sequencesthat is useful in establishing homology varies with the nucleic acid andprotein at issue, but as little as 25% sequence identity is routinelyused to establish homology. Higher levels of sequence identity, e.g.,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more can also be usedto establish homology. Methods for determining sequence identitypercentages (e.g., BLASTP and BLASTN using default parameters) aredescribed herein and are generally available.

The term “subject” refers to any animal (e.g., a mammal), including, butnot limited to, humans, non-human primates, canines, felines, rodents,and the like, which is to be the recipient of a particular treatment.Typically, the terms “subject” and “patient” are used interchangeablyherein in reference to a human subject.

The terms “effective amount” or “therapeutically effective amount” or“therapeutic effect” refer to an amount of a therapeutic effective to“treat” a disease or disorder in a subject or mammal. Thetherapeutically effective amount of a drug has a therapeutic effect andas such can prevent the development of a disease or disorder; slow downthe development of a disease or disorder; slow down the progression of adisease or disorder; relieve to some extent one or more of the symptomsassociated with a disease or disorder; reduce morbidity and mortality;improve quality of life; or a combination of such effects.

The terms “treating” or “treatment” or “to treat” or “alleviating” or“to alleviate” refer to both (1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder; and (2) prophylactic or preventativemeasures that prevent or slow the development of a targeted pathologiccondition or disorder. Thus those in need of treatment include thosealready with the disorder; those prone to have the disorder; and thosein whom the disorder is to be prevented.

“Pharmaceutically acceptable” refers to a generally non-toxic, inert,and/or physiologically compatible composition or component of acomposition.

A “pharmaceutical excipient” or “excipient” comprises a material such asan adjuvant, a carrier, pH-adjusting and buffering agents, tonicityadjusting agents, wetting agents, preservatives, and the like. A“pharmaceutical excipient” is an excipient which is pharmaceuticallyacceptable.

II. Neoantigens and Uses Thereof

One of the critical barriers to developing curative and tumor-specificimmunotherapy is the identification and selection of highly specific andrestricted tumor antigens to avoid autoimmunity. Tumor neoantigens,which arise as a result of genetic change (e.g., inversions,translocations, deletions, missense mutations, splice site mutations,etc.) within malignant cells, represent the most tumor-specific class ofantigens. Neoantigens have rarely been used in cancer vaccine orimmunogenic compositions due to technical difficulties in identifyingthem, selecting optimized antigens, and producing neoantigens for use ina vaccine or immunogenic composition. These problems may be addressedby: identifying mutations in neoplasias/tumors which are present at theDNA level in tumor but not in matched germline samples from a highproportion of subjects having cancer; analyzing the identified mutationswith one or more peptide-MHC binding prediction algorithms to generate aplurality of neoantigen T cell epitopes that are expressed within theneoplasia/tumor and that bind to a high proportion of patient HLAalleles; and synthesizing the plurality of neoantigenic peptidesselected from the sets of all neoantigen peptides and predicted bindingpeptides for use in a cancer vaccine or immunogenic composition suitablefor treating a high proportion of subjects having cancer.

For example, translating peptide sequencing information into atherapeutic vaccine may include prediction of mutated peptides that canbind to HLA molecules of a high proportion of individuals. Efficientlychoosing which particular mutations to utilize as immunogen requires theability to predict which mutated peptides would efficiently bind to ahigh proportion of patient's HLA alleles. Recently, neural network basedlearning approaches with validated binding and non-binding peptides haveadvanced the accuracy of prediction algorithms for the major HLA-A and-B alleles. However, even using advanced neural network-based algorithmsto encode HLA-peptide binding rules, several factors limit the power topredict peptides presented on HLA alleles.

Another example of translating peptide sequencing information into atherapeutic vaccine may include formulating the drug as a multi-epitopevaccine of long peptides. Targeting as many mutated epitopes aspractically possible takes advantage of the enormous capacity of theimmune system, prevents the opportunity for immunological escape bydown-modulation of an immune targeted gene product, and compensates forthe known inaccuracy of epitope prediction approaches. Syntheticpeptides provide a useful means to prepare multiple immunogensefficiently and to rapidly translate identification of mutant epitopesto an effective vaccine. Peptides can be readily synthesized chemicallyand easily purified utilizing reagents free of contaminating bacteria oranimal substances. The small size allows a clear focus on the mutatedregion of the protein and also reduces irrelevant antigenic competitionfrom other components (non-mutated protein or viral vector antigens).

Yet another example of translating peptide sequencing information into atherapeutic vaccine may include a combination with a strong vaccineadjuvant. Effective vaccines may require a strong adjuvant to initiatean immune response. For example, poly-ICLC, an agonist of TLR3 and theRNA helicase-domains of MDA5 and RIG3, has shown several desirableproperties for a vaccine adjuvant. These properties include theinduction of local and systemic activation of immune cells in vivo,production of stimulatory chemokines and cytokines, and stimulation ofantigen-presentation by DCs. Furthermore, poly-ICLC can induce durableCD4⁺ and CD8⁺ responses in humans. Importantly, striking similarities inthe upregulation of transcriptional and signal transduction pathwayswere seen in subjects vaccinated with poly-ICLC and in volunteers whohad received the highly effective, replication-competent yellow fevervaccine. Furthermore, >90% of ovarian carcinoma patients immunized withpoly-ICLC in combination with a NYESO-1 peptide vaccine (in addition toMontanide) showed induction of CD4⁺ and CD8⁺ T cell, as well as antibodyresponses to the peptide in a recent phase 1 study. At the same time,poly-ICLC has been extensively tested in more than 25 clinical trials todate and exhibited a relatively benign toxicity profile.

In some aspects, provided herein is a composition comprising: a firstpeptide comprising a first neoepitope of a protein and a second peptidecomprising a second neoepitope of the same protein, a polynucleotideencoding the first peptide and the second peptide, one or more APCscomprising the first peptide and the second peptide, or a first T cellreceptor (TCR) specific for the first neoepitope in complex with an HLAprotein and a second TCR specific for the second neoepitope in complexwith an HLA protein; wherein the first peptide is different from thesecond peptide, and wherein the first neoepitope comprises a mutationand the second neoepitope comprises the same mutation.

In some aspects, provided herein is a composition comprising: a firstpeptide comprising a first neoepitope of a region of a protein and asecond peptide comprising a second neoepitope of the region of the sameprotein, wherein the first neoepitope and the second neoepitope compriseat least one amino acid of the region that is the same, a polynucleotideencoding the first peptide and the second peptide, on or more APCscomprising the first peptide and the second peptide, or a first T cellreceptor (TCR) specific for the first neoepitope in complex with an HLAprotein and a second TCR specific for the second neoepitope in complexwith an HLA protein; wherein the first peptide is different from thesecond peptide, and wherein the first neoepitope comprises a firstmutation and the second neoepitope comprises a second mutation.

In some embodiments, the first mutation and the second mutation are thesame. In some embodiments, the first peptide and the second peptide aredifferent molecules. In some embodiments, the first neoepitope comprisesa first neoepitope of a region of the same protein, wherein the secondneoepitope comprises a second neoepitope of the region of the sameprotein. In some embodiments, the first neoepitope and the secondneoepitope comprise at least one amino acid of the region that is thesame. In some embodiments, the region of the protein comprises at least9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350,400, 450, 500, 600, 700, 800, 900, or 1,000 contiguous amino acids ofthe protein. In some embodiments, the region of the protein comprises atmost 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350,400, 450, 500, 600, 700, 800, 900, or 1,000 contiguous amino acids ofthe protein. In some embodiments, the first neoepitope binds to a classI HLA protein to form a class I HLA-peptide complex. In someembodiments, the second neoepitope binds to a class II HLA protein toform a class II HLA-peptide complex. In some embodiments, the secondneoepitope binds to a class I HLA protein to form a class I HLA-peptidecomplex. In some embodiments, the first neoepitope binds to a class IIHLA protein to form a class II HLA-peptide complex. In some embodiments,the first neoepitope is a first neoepitope peptide processed from thefirst peptide and/or the second neoepitope is a second neoepitopepeptide processed from the second peptide. In some embodiments, thefirst neoepitope is shorter in length than first peptide and/or thesecond neoepitope is shorter in length than second peptide. In someembodiments, the first neoepitope peptide is processed by an antigenpresenting cell (APC) comprising the first peptide and/or the secondneoepitope peptide is processed by an APC comprising the second peptide.In some embodiments, the first neoepitope activates CD8⁺ T cells. Insome embodiments, the second neoepitope activates CD4⁺ T cells. In someembodiments, the second neoepitope activates CD8⁺ T cells. In someembodiments, the first neoepitope activates CD4⁺ T cells. In someembodiments, a TCR of a CD4⁺ T cell binds to a class II HLA-peptidecomplex comprising the first or second peptide. In some embodiments, aTCR of a CD8⁺ T cell binds to a class I HLA-peptide complex comprisingthe first or second peptide. In some embodiments, a TCR of a CD4⁺ T cellbinds to a class I HLA-peptide complex comprising the first or secondpeptide. In some embodiments, a TCR of a CD8⁺ T cell binds to a class IIHLA-peptide complex comprising the first or second peptide. In someembodiments, the one or more APCs comprise a first APC comprising thefirst peptide and a second APC comprising the second peptide. In someembodiments, the mutation is selected from the group consisting of apoint mutation, a splice-site mutation, a frameshift mutation, aread-through mutation, a gene fusion mutation and any combinationthereof. In some embodiments, the first neoepitope and the secondneoepitope comprises a sequence encoded by a gene of Table 1 or 2. Insome embodiments, the protein is encoded by a gene of Table 1 or 2. Insome embodiments, the mutation is a mutation of column 2 of Table 1 or2. In some embodiments, the mutation is a mutation of column 1 of Tables3 to 14. In some embodiments, the protein is KRAS. In some embodiments,a single polypeptide comprises the first peptide and the second peptide,or a single polynucleotide encodes the first peptide and the secondpeptide. In some embodiments, the first peptide and the second peptideare encoded by a sequence transcribed from a same transcription startsite. In some embodiments, the first peptide is encoded by a sequencetranscribed from a first transcription start site and the second peptideis encoded by a sequence transcribed from a second transcription startsite. In some embodiments, the single polypeptide has a length of atleast 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 40; 50; 60;70; 80; 90; 100; 150; 200; 250; 300; 350; 400; 450; 500; 600; 700; 800;900; 1,000; 1,500; 2,000; 2,500; 3,000; 4,000; 5,000; 7,500; or 10,000amino acids. In some embodiments, the polypeptide comprises a firstsequence with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99%, sequence identity to a first corresponding wild-typesequence; and a second sequence with at least 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to acorresponding second wild-type sequence. In some embodiments, thepolypeptide comprises a first sequence of at least 8 or 9 contiguousamino acids with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99%, sequence identity to a corresponding first wild-typesequence; and a second sequence of at least 16 or 17 contiguous aminoacids with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99%, sequence identity to a corresponding second wild-typesequence. In some embodiments, the second peptide is longer than thefirst peptide In some embodiments, the first peptide is longer than thesecond peptide. In some embodiments, the first peptide has a length ofat least 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24;25; 26; 27; 28; 29; 30; 40; 50; 60; 70; 80; 90; 100; 150; 200; 250; 300;350; 400; 450; 500; 600; 700; 800; 900; 1,000; 1,500; 2,000; 2,500;3,000; 4,000; 5,000; 7,500; or 10,000 amino acids. In some embodiments,the second peptide has a length of at least 17; 18; 19; 20; 21; 22; 23;24; 25; 26; 27; 28; 29; 30; 40; 50; 60; 70; 80; 90; 100; 150; 200; 250;300; 350; 400; 450; 500; 600; 700; 800; 900; 1,000; 1,500; 2,000; 2,500;3,000; 4,000; 5,000; 7,500; or 10,000 amino acids. In some embodiments,the first peptide comprises a sequence of at least 9 contiguous aminoacids with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity to a corresponding wild-type sequence. In someembodiments, the second peptide comprises a sequence of at least 17contiguous amino acids with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity to a corresponding wild-typesequence. In some embodiments, the second neoepitope is longer than thefirst neoepitope. In some embodiments, the first neoepitope has a lengthof at least 8 amino acids. In some embodiments, the first neoepitope hasa length of from 8 to 12 amino acids. In some embodiments, the firstneoepitope comprises a sequence of at least 8 contiguous amino acids,wherein at least 2 of the 8 contiguous amino acids are different atcorresponding positions of a wild-type sequence. In some embodiments,the second neoepitope has a length of at least 16 amino acids. In someembodiments, the second neoepitope has a length of from 16 to 25 aminoacids. In some embodiments, the second neoepitope comprises a sequenceof at least 16 contiguous amino acids, wherein at least 2 of the 16contiguous amino acids are different at corresponding positions of awild-type sequence.

In some embodiments, the first peptide comprises at least one anadditional mutation. In some embodiments, one or more of the at leastone additional mutation is not a mutation in the first neoepitope. Insome embodiments, one or more of the at least one additional mutation isa mutation in the first neoepitope. In some embodiments, the secondpeptide comprises at least one additional mutation. In some embodiments,one or more of the at least one additional mutation is not a mutation inthe second neoepitope. In some embodiments, one or more of the at leastone additional mutation is a mutation in the second neoepitope. In someembodiments, the first peptide, the second peptide or both comprise atleast one flanking sequence, wherein the at least one flanking sequenceis upstream or downstream of the neoepitope. In some embodiments, the atleast one flanking sequence has at least 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to acorresponding wild-type sequence. In some embodiments, the at least oneflanking sequence comprises a non-wild-type sequence. In someembodiments, the at least one flanking sequence is a N-terminus flankingsequence. In some embodiments, the at least one flanking sequence is aC-terminus flanking sequence. In some embodiments, the at least oneflanking sequence of the first peptide has at least 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to theat least one flanking sequence of the second peptide. In someembodiments, the at least one flanking region of the first peptide isdifferent from the at least one flanking region of the second peptide.In some embodiments, the at least one flanking residue comprises themutation. In some embodiments, the first neoepitope, the secondneoepitope or both comprises at least one anchor residue. In someembodiments, the at least one anchor residue of the first neoepitope isat a canonical anchor position. In some embodiments, the at least oneanchor residue of the first neoepitope is at a non-canonical anchorposition. In some embodiments, the at least one anchor residue of thesecond neoepitope is at a canonical anchor position. In someembodiments, the at least one anchor residue of the second neoepitope isat a non-canonical anchor position. In some embodiments, the at leastone anchor residue of the first neoepitope is different from the atleast one anchor residue of the second neoepitope. In some embodiments,the at least one anchor residue is a wild-type residue. In someembodiments, the at least one anchor residue is a substitution. In someembodiments, the first neoepitope and/or the second neoepitope binds toan HLA protein with a greater affinity than a corresponding neoepitopewithout the substitution. In some embodiments, the first neoepitopeand/or the second neoepitope binds to an HLA protein with a greateraffinity than a corresponding wild-type sequence without thesubstitution. In some embodiments, at least one anchor residue does notcomprise the mutation. In some embodiments, the first neoepitope, thesecond neoepitope or both comprise at least one anchor residue flankingregion. In some embodiments, the neoepitope comprises at least oneanchor residue. In some embodiments, the at least one anchor residuescomprises at least two anchor residues. In some embodiments, the atleast two anchor residues are separated by a separation regioncomprising at least 1 amino acid. In some embodiments, the at least oneanchor residue flanking region is not within the separation region. Insome embodiments, the at least one anchor residue flanking region isupstream of a N-terminal anchor residue of the at least two anchorresidues downstream of a C-terminal anchor residue of the at least twoanchor residue both (a) and (b).

In some embodiments, composition comprises an adjuvant. In someembodiments, the composition comprises one or more additional peptides,wherein the one or more additional peptides comprise a third neoepitope.In some embodiments, the first and/or second neoepitope binds to an HLAprotein with a greater affinity than a corresponding wild-type sequence.In some embodiments, the first and/or second neoepitope binds to an HLAprotein with a K_(D) or an IC₅₀ less than 1000 nM, 900 nM, 800 nM, 700nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM or 10 nM. Insome embodiments, the first and/or second neoepitope binds to an HLAclass I protein with a K_(D) or an IC₅₀ less than 1000 nM, 900 nM, 800nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM or 10nM. In some embodiments, the first and/or second neoepitope binds to anHLA class II protein with a K_(D) or an IC₅₀ less than 1000 nM, 900 nM,800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM or10 nM. In some embodiments, the first and/or second neoepitope binds toa protein encoded by an HLA allele expressed by a subject. In someembodiments, the mutation is not present in non-cancer cells of asubject. In some embodiments, the first and/or second neoepitope isencoded by a gene or an expressed gene of a subject's cancer cells. Insome embodiments, the composition comprises a first T cell comprisingthe first TCR. In some embodiments, the composition comprises a second Tcell comprising the second TCR. In some embodiments, the first TCRcomprises a non-native intracellular domain and/or the second TCRcomprises a non-native intracellular domain. In some embodiments, thefirst TCR is a soluble TCR and/or the second TCR is a soluble TCR. Insome embodiments, the first and/or second T cell is a cytotoxic T cell.In some embodiments, the first and/or second T cell is a gamma delta Tcell. In some embodiments, the first and/or second T cell is a helper Tcell. In some embodiments, the first T cell is a T cell stimulated,expanded or induced with the first neoepitope and/or the second T cellis a T cell stimulated, expanded or induced with the second neoepitope.In some embodiments, the first and/or second T cell is an autologous Tcell. In some embodiments, the first and/or second T cell is anallogenic T cell. In some embodiments, the first and/or second T cell isan engineered T cell. In some embodiments, the first and/or second Tcell is a T cell of a cell line. In some embodiments, the first and/orsecond TCR binds to an HLA-peptide complex with a K_(D) or an IC₅₀ ofless than 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150nM, 100 nM, 50 nM, 25 nM or 10 nM. In some aspects, provided herein is avector comprising a polynucleotide encoding a first and a second peptidedescribed herein. In some embodiments, the polynucleotide is operablylinked to a promoter. In some embodiments, the vector is aself-amplifying RNA replicon, plasmid, phage, transposon, cosmid, virus,or virion. In some embodiments, the vector is a viral vector. In someembodiments, the vector is derived from a retrovirus, lentivirus,adenovirus, adeno-associated virus, herpes virus, pox virus, alphavirus, vaccina virus, hepatitis B virus, human papillomavirus or apseudotype thereof. In some embodiments, the vector is a non-viralvector. In some embodiments, the non-viral vector is a nanoparticle, acationic lipid, a cationic polymer, a metallic nanopolymer, a nanorod, aliposome, a micelle, a microbubble, a cell-penetrating peptide, or aliposphere.

In some aspects, provided herein is a pharmaceutical compositioncomprising: a composition described herein, or a vector describedherein; and a pharmaceutically acceptable excipient.

In some embodiments, the plurality of cells is autologous cells. In someembodiments, the plurality of APC cells is autologous cells. In someembodiments, the plurality of T cells is autologous cells. In someembodiments, the pharmaceutical composition further comprises animmunomodulatory agent or an adjuvant. In some embodiments, theimmunomodulatory agent is a cytokine. In some embodiments, the adjuvantis Hiltonol.

In some aspects, provided herein is a method of treating cancer, themethod comprising administering to a subject in need thereof apharmaceutical composition described herein.

In some aspects, provided herein is a method of preventing resistance toa cancer therapy, the method comprising administering to a subject inneed thereof a pharmaceutical composition described herein.

In some aspects, provided herein is a method of inducing an immuneresponse, the method comprising administering to a subject in needthereof a pharmaceutical composition described herein.

In some embodiments, the immune response is a humoral response. In someembodiments, the first peptide and the second peptide are administeredsimultaneously, separately or sequentially. In some embodiments, thefirst peptide is sequentially administered after the second peptide. Insome embodiments, the second peptide is sequentially administered afterthe first peptide. In some embodiments, the first peptide issequentially administered after a time period sufficient for the secondpeptide to activate the T cells. In some embodiments, the second peptideis sequentially administered after a time period sufficient for thefirst peptide to activate the T cells. In some embodiments, the firstpeptide is sequentially administered after the second peptide torestimulate the T cells. In some embodiments, the second peptide issequentially administered after the first peptide to restimulate the Tcells. In some embodiments, the first peptide is administered tostimulate the T cells and the second peptide is administered after thefirst peptide to restimulate the T cells. In some embodiments, thesecond peptide is administered to stimulate the T cells and the firstpeptide is administered after the second peptide to restimulate the Tcells. In some embodiments, the subject has cancer, wherein the canceris selected from the group consisting of melanoma, ovarian cancer, lungcancer, prostate cancer, breast cancer, colorectal cancer, endometrialcancer, and chronic lymphocytic leukemia (CLL). In some embodiments, thesubject has a breast cancer that is resistant to anti-estrogen therapy.In some embodiments, the breast cancer expresses an estrogen receptorwith a mutation. In some embodiments, the subject has a CLL that isresistant to ibrutinib therapy. In some embodiments, the CLL expresses aBruton tyrosine kinase with a mutation, such as a C481S mutation. Insome embodiments, the subject has a lung cancer that is resistant to atyrosine kinase inhibitor. In some embodiments, the lung cancerexpresses an epidermal growth factor receptor (EGFR) with a mutation,such as a T790M, L792F, or C797S mutation. In some embodiments, theplurality of APC cells comprising the first peptide and the plurality ofAPC cells comprising the second peptide are administered simultaneously,separately or sequentially. In some embodiments, the plurality of Tcells comprising the first TCR and the plurality of T cells comprisingthe second TCR are administered simultaneously, separately orsequentially. In some embodiments, the method further comprisesadministering at least one additional therapeutic agent or modality. Insome embodiments, the at least one additional therapeutic agent ormodality is surgery, a checkpoint inhibitor, an antibody or fragmentthereof, a chemotherapeutic agent, radiation, a vaccine, a smallmolecule, a T cell, a vector, and APC, a polynucleotide, an oncolyticvirus or any combination thereof. In some embodiments, the at least oneadditional therapeutic agent is an anti-PD-1 agent and anti-PD-L1 agent,an anti-CTLA-4 agent, or an anti-CD40 agent. In some embodiments, theadditional therapeutic agent is administered before, simultaneously, orafter administering a pharmaceutical composition according describedherein.

III. Peptides

In aspects, the present disclosure provides isolated peptides thatcomprise a tumor specific mutation from Table 1 to 14. These peptidesand polypeptides are referred to herein as “neoantigenic peptides” or“neoantigenic polypeptides”. The polypeptides or peptides can be avariety of lengths, either in their neutral (uncharged) forms or informs which are salts, and either free of modifications such asglycosylation, side chain oxidation, or phosphorylation or containingthese modifications, subject to the condition that the modification notdestroy the biological activity of the polypeptides as herein described.

TABLE 1 Exemplary Mutation Peptides Protein Sequence (HLA alleleExemplary Gene Change Context example(s)) DiseasesTABLE 1A POINT MUTATION ¹ ABL1 E255K VADGLITTLHYPAPKR GQYGKVYEG (A02.01)Chronic myeloid NKPTVYGVSPNYDKW GQYGKVYEGV (A02.01) leukemia (CML),EMERTDITMKHKLGG KLGGGQYGK (A03.01) Acute lymphocytic GQYGKVYEGVWKKYKLGGGQYGKV (A02.01) leukemia (ALL), SLTVAVKTLKEDTME KVYEGVWKK(A02.01,Gastrointestinal VEEFLKEAAVMKEIK A03.01) stromal tumors HPNLVQLLGVCKVYEGVWKKY (A03.01) (GIST) QYGKVYEGV (A24.02) QYGKVYEGVW (A24.02) ABL1E255V VADGLITTLHYPAPKR GQYGVVYEG (A02.01) Chronic myeloidNKPTVYGVSPNYDKW GQYGVVYEGV (A02.01) leukemia (CML), EMERTDITMKHKLGGKLGGGQYGV (A02.01) Acute lymphocytic GQYGVVYEGVWKKY KLGGGQYGVV (A02.01)leukemia (ALL), SLTVAVKTLKEDTME QYGVVYEGV (A24.02) GastrointestinalVEEFLKEAAVMKEIK QYGVVYEGVW (A24.02) stromal tumors HPNLVQLLGVCVVYEGVWKK (A02.01, (GIST) A03.01) VVYEGVWKKY (A03.01) ABL1 M351TLLGVCTREPPFYIITEF ATQISSATEY (AO 1.01) Chronic myeloid MTYGNLLDYLRECNRISSATEYLEK (A03.01) leukemia (CML), QEVNAVVLLYMATQI SSATEYLEK (A03.01)Acute lymphocytic SSATEYLEKKNFIHRD TQISSATEYL (A02.01) leukemia (ALL),LAARNCLVGENHLVK YMATQISSAT (A02.01) Gastrointestinal VADFGLSRLMTGDTYstromal tumors TAHAGAKF (GIST) ABL1 T3151 SLTVAVKTLKEDTMEFYIIIEFMTY (A24.02) Chronic myeloid VEEFLKEAAVMKEIK IIEFMTYGNL (A02.01)leukemia (CML), HPNLVQLLGVCTREPP IIIEFMTYG (A02.01) Acute lymphocyticFYIIIEFMTYGNLLDYL IIIEFMTYGN (A02.01) leukemia (ALL), RECNRQEVNAVVLLYYIIIEFMTYG (A02.01) Gastrointestinal MATQISSAMEYLEKK stromal tumorsNFIHRDLA (GIST) ABL1 Y253H STVADGLITTLHYPAP GQHGEVYEGV (A02.01)Chronic myeloid KRNKPTVYGVSPNYD KLGGGQHGEV (A02.01) leukemia (CML),KWEMERTDITMKHKL Acute lymphocytic GGGQHGEVYEGVWK leukemia (ALL),KYSLTVAVKTLKEDT Gastrointestinal MEVEEFLKEAAVMKE stromal tumorsIKHPNLVQLLG (GIST) ALK GI269A SSLAMLDLLHVARDI KIADFGMAR (A03.01) NSCLCACGCQYLEENHFIHR RVAKIADFGM (A02.01, DIAARNCLLTCPGPGR B07.02)VAKIADFGMARDIYR ASYYRKGGCAMLPVK WMPPEAFMEGIFTSKT DTWSFGVLL ALK L1I96MQVAVKTLPEVCSEQD FILMELMAGG (A02.01) NSCLC ELDFLMEALIISKFNHILMELMAGG (A02.01) QNIVRCIGVSLQSLPRF ILMELMAGGD (A02.01) ILMELMAGGDLKSFLMELMAGGDL (A02.01) LRETRPRPSQPSSLAML LPRFILMEL (B07.02, DLLHVARDIACGCQY B08.01) LEENHFI LPRFILMELM (B07.02) LQSLPRFILM (A02.01,B08.01) SLPRFILMEL (A02.01, A24.02, B07.02, B08.01) BRAF V600EMIKLIDIARQTAQGMD LATEKSRWS (A02.01, CRC, GBM, KIRP, YLHAKSIIHRDLKSNNB08.01) LUAD, SKCM, IFLHEDLTVKIGDFGL LATEKSRWSG (A02.01, THCAATEKSRWSGSHQFEQ B08.01) LSGSILWMAPEVIRMQ DKNPYSFQSDVYAFGI VLYELM BTKC481S MIKEGSMSEDEFIEEA EYMANGSLL (A24.02) CLL KVMMNLSHEKLVQLMANGSLLNY (A01.01, YGVCTKQRPIFIITEY A03.01, A11.01) MANGSLLNYLREMRHMANGSLLNYL (A02.01, RFQTQQLLEMCKDVC B07.02, B08.01) EAMEYLESKQFLHRDSLLNYLREM (A02.01, LAARNCLVND B07.02, B08.01) YMANGSLLN (A02.01)YMANGSLLNY (A01.01, A03.01, A11.01) EEF1B2 S43G MGFGDLKSPAGLQVLGPPPADLCHAL (B07.02) BLCA, KIRP, NDYLADKSYIEGYVPS PRAD, SKCMQADVAVFEAVSGPPP ADLCHALRWYNHIKS YEKEKASLPGVKKAL GKYGPADVEDTTGSG AT EGFRS492R SLNITSLGLRSLKEISD IIRNRGENSCK (A03.01) CRC GDVIISGNKNLCYANTINWKKLFGTSGQKTKI IRNRGENSCKATGQV CHALCSPEGCWGPEP RDCVSCRNVSRGREC VDKCNLLEGFR T790M IPVAIK.ELREATSPKA CLTSTVQLIM (A01.01, NSCLC, PRADNKEILDEAYVMASVD A02.01) NPHVCRLLGICLTSTV 1MQLMPFGC (A02.01)QLIMQLMPFGCLLDY IMQLMPFGCL (A02.01, VREHKDNIGSQYLLN A24.02, B08.01)WCVQ1AKGMNYLED L1MQLMPFG (A02.01) RRLVHRDLAA LIMQLMPFGC (A02.01)LTSTVQL1M (A01.01) MQLMPFGCL (A02.01, B07.02, B08.01)MQLMPFGCLL (A02.01, A24.02, B08.01) QLIMQLMPF (A02.01, A24.02, B08.01)QLIMQLMPFG (A02.01) STVQLIMQL (A02.01) VQLIMQLMPF (A02.01,A24.02, B08.01) ERBB3 VI04M ERCEVVMGNLEIVLT CRC, Stomach GHNADLSFLQWIREVCancer TGYVLVAMNEFSTLP LPNLRMVRGTQVYDG KFAIFVMLNYNTNSSH ALRQLRLTQLTEILSGGVYIEKNDK ESR1 D538G HLMAKAGLTLQQQH GLLLEMLDA (A02.01) Breast CancerQRLAQLLLILSHIRHM LYGLLLEML (A24.02) SNKGMEHLYSMKCK NVVPLYGLL (A02.01)NVVPLYGLLLEMLDA PLYGLLLEM (A02.01) HRLHAPTSRGGASVE PLYGLLLEML (A02.01,ETDQSHLATAGSTSSH A24.02) SLQKYYITGEA VPLYGLLLEM (B07.02)VVPLYGLLL (A02.01, A24.02) ESR1 S463P NQGKCVEGMVEIFDM FLPSTLKSL Breast Cancer LLATSSRFRMMNLQG (A02.01,  EEFVCLKSIILLNSGVY A24.02,TFLPSTLKSLEEKDHIH B08.01) RVLDKITDTLIHLMAK GVYTFLPST (A02.01)AGLTLQQQHQRLAQL GVYTFLPSTL (A02.01, LLILSH A24.02) TFLPSTLKSL (A24.02)VYTFLPSTL (A24.02) YTFLPSTLK (A03.01) ESR1 Y537C IHLMAKAGLTLQQQHNVVPLCDLL (A02.01) Breast Cancer QRLAQLLLILSHIRHM NVVPLCDLLL (A02.01)SNKGMEHLYSMKCK PLCDLLLEM (A02.01) NVVPLCDLLLEMLDA PLCDLLLEML (A02.01)HRLHAPTSRGGASVE VPLCDLLLEM (B07.02) ETDQSHLATAGSTSSH VVPLCDLLL (A02.01,SLQKYYITGE A24.02) ESR1 Y537N IHLMAKAGLTLQQQH NVVPLNDLL (A02.01)Breast Cancer QRLAQLLLILSHIRHM NVVPLNDLLL (A02.01) SNKGMEHLYSMKCKPLNDLLLEM (A02.01) NVVPLNDLLLEMLDA PLNDLLLEML (A02.01) HRLHAPTSRGGASVEVPLNDLLLEM (B07.02) ETDQSHLATAGSTSSH SLQKYYITGE ESR1 Y337SIHLMAKAGLTLQQQH NVVPLSDLL (A02.01) Breast Cancer QRLAQLLLILSHIRHMNVVPLSDLLL (A02.01) SNKGMEHLYSMKCK PLSDLLLEM (A02.01) NVVPLSDLLLEMLDAPLSDLLLEML (A02.01) HRLHAPTSRGGASVE VPLSDLLLEM (B07.02) ETDQSHLATAGSTSSHVVPLSDLLL (A02.01, SLQKYYITGE A24.02) FGFR3 S249C HRIGGIKLRHQQWSLVLERCPHRPI (A02.01, BLCA, HNSC, VMESVVPSDRGNYTC B08.01) KIRP, LUSCVVENKFGSIRQTYTLD YTLDVLERC (A02.01) VLERCPHRPILQAGLP ANQTAVLGSDVEFHCKVYSDAQPHIQWLKH VEVNGSKVG FRG1B L52S AVKLSDSRIALKSGYG FQNGKMALS (A02.01)GBM,KIRP, KYLGINSDELVGHSD PRAD, SKCM AIGPREQWEPVFQNG KMALSASNSCFIRCNEAGDIEAKSKTAGEEE MIKIRSCAEKETKKKD DIPEEDKG HER2 V777L GSGAFGTVYKGIWIPDVMAGLGSPYV (A02.01, BRCA (Resistance) GENVKIPVAIKVLREN A03.01)TSPKANKEILDEAYV MAGLGSPYVSRLLGIC LTSTVQLVTQLMPYG CLLDHVRENRGRLGSQDLLNWCM IDH1 R132H RVEEFKLKQMWKSPN KPIIIGHHA (B07.02) BLCA, GBM,GTIRNILGGTVFREAII PRAD CKNIPRLVSGWVKPIII GHHAYGDQYRATDF VVPGPGKVEITYTPSDGTQKVTYLVHNFEEG GGVAMGM IDH1 R132C RVEEFKLKQMWKSPN KPIIIGCHA (B07.02)BLCA, GBM, GTIRNILGGTVFREAII PRAD CKNIPRLVSGWVKPIII GCHAYGDQYRATDFVVPGPGKVEITYTPSDG TQKVTYLVHNFEEGG GVAMGM IDH1 RI32G RVEEFKLKQMWKSPNKPIIIGGHA (B07.02) BLCA, BRCA, GTIRNILGGTVFREAII CRC, GBM,CKNIPRLVSGWVKPIII HNSC, LUAD, GGHAYGDQYRATDF PAAD, PRAD,VVPGPGKVEITYTPSD UCEC GTQKVTYLVHNFEEG GGVAMGM IDH1 R132S RVEEFKLKQMWKSPNKPII1GSHA (B07.02) BLCA, BRCA, GTIRNILGGTVFREAII GBM, HNSC,CKNIPRLVSGWVKPIII LIHC, LUAD, GSHAYGDQYRATDFV LUSC, PAAD,VPGPGKVEITYTPSDG SKCM, UCEC TQKVTYLVHNFEEGG GVAMGM KIT T670IVAVKMLKPSAHLTER I1EYCCYGDL (A02.01) Gastrointestinal EALMSELKVLSYLGNTIGGPTLVII (A02.01) stromal tumors HMNIVNLLGACTIGGP VIIEYCCYG (A02.01)(GIST) TLVIIEYCCYGDLLNF LRRKRDSFICSKQEDH AEAALYKNLLHSKES SCSDSTNE KITV654A VEATAYGLIKSDAAM HMNIANLLGA (A02.01) GastrointestinalTVAVKMLKPSAHLTE IANLLGACTI (A02.01) stromal tumors REALMSELKVLSYLGMNIANLLGA (A02.01) (GIST) NHMNIANLLGACTIG YLGNHMNIA (A02.01,GPTLVITEYCCYGDLL B08.01) NFLRRKRDSFICSKQE YLGNHMNIAN (A02.01) DHAEAALYKMEK C121S ISELGAGNGGVVFKVS VLHESNSPY (A03.01) Melanoma HKPSGLVMARKLIHLVLHESNSPYI (A02.01) EIKPAIRNQIIRELQVL HESNSPYIVGFYGAFY SDGEISICMEHMDGGSLDQVLKKAGRIPEQIL GKVSI MEK P124L LGAGNGGVVFKVSHK LQVLHECNSL (A02.01,Melanoma PSGLVMARKLIHLEIK B08.01) PAIRNQIIRELQVLHEC LYIVGFYGAF (A24.02)NSLYIVGFYGAFYSDG NSLYIVGFY (AO 1.01) EISICMEHMDGGSLDQ QVLHECNSL (A02.01,VLKKAGRIPEQILGKV B08.01) SIAVI SLYIVGFYG (A02.01) SLYIVGFYGA (A02.01)VLHECNSLY (A03.01) VLHECNSLYI (A02.01, A03.01) MYC E39D MPLNVSFTNRNYDLDFYQQQQQSDL (A24.02) Lymphoid Cancer; YDSVQPYFYCDEEEN QQQSDLQPPA (A02.01)Burkitt Lymphoma FYQQQQQSDLQPPAPS QQSDLQPPA (A02.01) EDIWKKFELLPTPPLSPYQQQQQSDL (A02.01, SRRSGLCSPSYVAVTP B08.01) FSLRGDNDGG MYC P57SFTNRNYDLDYDSVQP FELLSTPPL (A02.01, Lymphoid Cancer YFYCDEEENFYQQQQB08.01) QSELQPPAPSEDIWKK LLSTPPLSPS (A02.01) FELLSTPPLSPSRRSGLCSPSYVAVTPFSLRGD NDGGGGSFSTADQLE MVTELLG MYC T58I TNRNYDLDYDSVQPYFELLPIPPL (A02.01) Neuroblastoma FYCDEEENFYQQQQQ IWKKFELLPI (A24.02)SELQPPAPSEDIWKKF LLPIPPLSPS (A02.01, ELLPIPPLSPSRRSGLC B07.02)SPSYVAVTPFSLRGDN LPIPPLSPS (B07.02) DGGGGSFSTADQLEM VTELLGG PDGFRa T674IVAVKMLKPTARSSEK IIEYCFYGDL (A02.01) Chronic QALMSELKIMTHLGPII1EYCFYG (A02.01) Eosinophilic HLNIVNLLGACTKSGP IYIIIEYCF (A24.02)Leukemia IYIIIEYCFYGDLVNYL IYIIIEYCFY (A24.02) HKNRDSFLSHHPEKPKYI1IEYCFYG (A02.01) KELDIFGLNPADESTR SYVILS PIK3CA E542KIEEHANWSVSREAGFS KITEQEKDFL (A02.01) BLCA, BRCA, YSHAGLSNRLARDNECESC, CRC,  LRENDKEQLKAISTRD GBM, PLSKITEQEKDFLWSH HNSC, K1RC,RHYCVTIPEILPKLLLS KIRP, LIHC, VKWNSRDEVAQMYC LUAD, LUSC, LVKDWPPPRAD, UCEC PIK3CA E545K HANWSVSREAGFSYS STRDPLSE1TK (A03.01) BLCA, BRCA,HAGLSNRLARDNELR DPLSEITK (A03.01) CESC, CRC, ENDKEQLKAISTRDPL GBM,SEITKQEKDFLWSHRH HNSC, KIRC, YCVTIPEILPKLLLSVK KIRP, LIHC,WNSRDEVAQMYCLV LUAD, LUSC, KDWPPiKP PRAD, SKCM, UCEC PIK3CA H1047RLFINLFSMMLGSGMPE BRCA, CESC, LQSFDDIAYIRKTLAL CRC, GBM, DKTEQEALEYFMKQMHNSC, LIHC, NDARHGGWTTKMDW LUAD, LUSC, IFHTIKQHALN PRAD, UCEC POLE P286RQRGGVITDEEETSKKI LPLKFRDAET  Colorectal ADQLDNIVDMREYDV (B07.02)adenocarcinoma; PYHIRLSIDIETTKLPL Uterine/ KFRDAETDQIMMISY EndometriumMIDGQGYLITNREIVS Adenocarcinoma; EDIEDFEFTPKPEYEGP Colorectal FCVFNadenocarcinoma, MSI +; Uterine/ Endometrium Adenocarcinoma, MSI +;Endometrioid carcinoma; Endometrium Serous carcinoma; EndometriumCarcinosarcoma- malignant mesodermal mixed tumor; Glioma;Astrocytoma; GBM PTEN RI30Q KFNCRVAQYPFEDHN QTGVMICAYL  BRCA, CESC,PPQLELIKPFCEDLDQ (A02.01) CRC, GBM, KiRC, WLSEDDNHVAAIHCK LUSC, UCECAGKGQTGVMICAYLL HRGKFLKAQEALDFY GEVRTRDKKGVTIPSQ RRYVYYYSY RAC1 P29SMQAIKCVVVGDGAV AFSGEYIPTV Melanoma GKTCLLISYTTNAFSG (A02.01,EYIPTVFDNYSANVM A24.02) VDGKPVNLGLWDTA GQEDYDRLRPLSYPQ TVGET TP53 G245S1RVEGNLRVEYLDDR SMNRRPILT BLCA, BRCA, NTFRHSVVVPYEPPEV (A02.01, B08.01)CRC, GBM, GSDCTTIHYNYMCNS YMCNSSCMGS HNSC, LUSC, SCMGSMNRRPILTIITL(A02.01) PAAD, PRAD EDSSGNLLGRNSFEVR VCACPGRDRRTEEEN LRKKGEP TP53 R175HTYSPALNKMFCQLAK BLCA, BRCA, TCPVQLWVDSTPPPGT CRC, GBM, RVRAMAIYKQSQHMTHNSC, LUAD, EVVRHCPHHERCSDS PAAD, PRAD, DGLAPPQHL1RVEGNL UCECRVEYLDDRNTFRHSV VVPYEPPEV TP53 R248Q EGNLRVEYLDDRNTF GMNQRPILT (A02.01)BLCA, BRCA, RHSVVVPYEPPEVGSD CRC, GBM, CTTIHYNYMCNSSCM HNSC, KIRC,GGMNQRPILTIITLEDS LIHC, LUSC, SGNLLGRNSFEVRVC PAAD, PRAD,ACPGRDRRTEEENLR UCEC KKGEPHHE TP53 R248W EGNLRVEYLDDRNTFGMNWRPILT (A02.01) BLCA, BRCA, RHSVVVPYEPPEVGSD CRC, GBM,CTTIHYNYMCNSSCM HNSC, LIHC, GGMNWRPILTIITLED LUSC, PAAD,SSGNLLGRNSFEVRVC SKCM, UCEC ACPGRDRRTEEENLR KKGEPHHE TP53 R273CPEVGSDCTTIHYNYM LLGRNSFEVC (A02.01) BLCA, BRCA, CNSSCMGGMNRRPILCRC, GBM, TIITLEDSSGNLLGRNS HNSC, LUSC, FEVCVCACPGRDRRT PAAD, UCECEEENLRKKGEPHHELP PGSTKRALPNNTSSSP QPKKKPLTABLE 1B MSI-ASSOCIATED FRAMESHIFTS 1 ACVR2 D96fs; +1 GVEPCYGDKDKRRHCMSI + CRC, MSI  A FATWKNISGSIEIVKQ Uterine/ GCWLDD1NCYDRTDC EndometriumVEKKRQP* Cancer, MSI  Stomach Cancer, Lynch syndrome ACVR2 D96fs; −1GVEPCYGDKDKRRHC ALKYIFVAV (A02.01, MSI + CRC, MSI  A FATWKNISGSIEIVKQB08.01) Uterine/ GCWLDDINCYDRTDC ALKYIFVAVR (A03.01) EndometriumVEKKTALKYIFVAVR AVRAICVMK (A03.01) Cancer, MSI  AICVMKSFLIFRRWKSAVRA1CVMKS (A03.01) Stomach Cancer, HSPLQIQLHLSHPITTS CVEKKTALK (A03.01)Lynch syndrome CSIPWCHLC* CVEKKTALKY (A01.01) CVMKSFLIF (A24.02, B08.01)CVMKSFLIFR (A03.01) FL1FRRWKS (A02.01,  B08.01) FRRWKSHSPL (B08.01)FVAVRAICV (A02.01, B08.01) FVAVRAICVM (B08.01) IQLHLSHPI (A02.01)KSFLIFRRWK (A03.01) KTALKYIFV (A02.01) KY1FVAVRAI (A24.02)RWKSHSPLQI (A24.02) TALKYIFVAV (A02.01, B08.01) VAVRAICVMK (A03.01)VMKSFLIFR (A03.01) VMKSFLIFRR (A03.01) YIFVAVRAI (A02.01) C150RFLI32fs; +1 TAEAVNVAIAAPPSEG ALFFFFFET (A02.01) MS1+ CRC, MSI  40EANAELCRYLSKVLE ALFFFFFETK (A03.01) Uterine/ LRKSDVVLDKVGLALAQAGVQWRSL (A02.01) Endomctrium  FFFFFETKSCSVAQAG CLANFCIFNR (A03.01)Cancer, MSI  VQWRSLGSLQPPPPGF CLSFLSSWDY (A01.01, Stomach Cancer,KLFSCLSFLSSWDYRR A03.01) Lynch syndrome MPPCLANFCIFNRDGVFFETKSCSV (B08.01) SPCWSGWS* FFFETKSCSV (A02.01) FKLFSCLSFL (A02.01)FLSSWDYRRM (A02.01) GFKLFSCLSF (A24.02) KLFSCLSFL (A02.01, A03.01)KLFSCLSFLS (A02.01, A03.01) LALFFFFFET (A02.01) LFFFFFETK (A03.01)LSFLSSWDY (A01.01) LSFLSSWDYR (A03.01) RMPPCLANF (A24.02)RRMPPCLANF (A24.02) SLQPPPPGFK (A03.01) VQWRSLGSL (A02.01) CNOT1L1544fs; +1 LSVIIFFFVYIWHWAL FFFSVIFST (A02.01) MSI + CRC, MSI PLILNNHHICLMSSIIL MSVCFFFFSV (A02.01) Uterine/ DCNSVRQSIMSVCFFFSVCFFFFSV (A02.01, Endometrium Cancer,  FSVIFSTRCLTDSRYPN B08.01)MSI Stomach Cancer, ICWFK* SVCFFFFSVI (A02.01) Lynch syndrome CNOT1L1544fs; −1 LSVIIFFFVYIWHWAL FFCYILNTMF (A24.02) MSI + CRC, MSI PLILNNHHICLMSSIIL MSVCFFFFCY (A01.01) Uterine/EndometriumDCNSVRQSIMSVCFFF SVCFFFFCYI (A02.01) Cancer, MSI  FCYILNTMFDR*Stomach Cancer, Lynch syndrome EIF2B3 A151fs; −1 VLVLSCDLITDVALHEKQWSSVTSL (A02.01) MSI + CRC, MSI  VVDLFRAYDASLAML VLWMPTSTV (A02.01)Uterine/ MRKGQDSIEPVPGQK Endometrium  GKKKQWSSVTSLEWT Cancer, MSI AQERGCSSWLMKQT Stomach Cancer, WMKSWSLRDPSYRSI Lynch syndromeLEYVSTRVLWMPTST V* EPHB2 K1020fs; −1 SIQVMRAQMNQIQSV ILIRKAMTV (A02.01)MSI + CRC, MSI  EGQPLARRPRATGRT Uterine/ KRCQPRDVTKKTCNS Endometrium NDGKKREWEKRKQIL Cancer, MSI  GGGGKYKEYFLKRILI Stomach Cancer,RKAMTVLAGDKKGL Lynch syndrome GRFMRCVQSETKAVS LQLPLGR* ESRP1 N512fs; +1LDFLGEFAIDIRTHGV MSI + CRC, MSI  HMVLNHQGRPSGDAF Uterine/IQMKSADRAFMAAQK Endometrium CHKKKHEGQIC* Cancer, MSI  Stomach Cancer,Lynch syndrome ESRP1 N512fs; −1 LDFLGEFATDIRTHGV MSI + CRC, MSI HMVLNHQGRPSGDAF Uterine/ IQMKSADRAFMAAQK Endometrium  CHKKT*Cancer, MSI  Stomach Cancer, Lynch syndrome FAM111 A273fs; −1GALCKDGRFRSDIGEF RMKVPLMK (A03.01) MSI + CRC, MSI  B EWKLKEGHKKIYGKQUterine/ SMVDEVSGKVLEMDI Endometrium  SKKKHYNRKISIKKLN Cancer, MSI RMKVPLMKLITRV* Stomach Cancer, Lynch syndrome GBP3 T585fs; −1RERAQLLEEQEKTLTS TLKKKPRDI (B08.01) MSI + CRC, MSI  KLQEQARVLKERCQGUterine/ ESTQLQNEIQKLQKTL Endometrium  KKKPRDICRIS* Cancer, MSI Stomach Cancer, Lynch syndrome JAK1 P861fs; +1 VNTLKEGKRLPCPPNCLIEGFEALLK (A03.01) MSI + CRC, MSI  PDEVYQLMRKCWEFQ Uterine/PSNRTSFQNLIEGFE Endometrium  ALLKTSN* Cancer, MSI  Stomach Cancer,Lynch syndrome JAKI K860fs; −1 CRPVTPSCKELADLM QQLKWTPHI (A02.01)MSI + CRC, MSI  TRCMNYDPNQRPFFR QLKWTPHILK (A03.01) Uterine/AIMRDINKLEEQNPDI IVSEKNQQLK (A03.01) Endometrium  VSEKNQQLKWTPHILQLKWTPHILK (A03.01) Cancer, MSI  KSAS* QQLKWTPHI (A24.02)Stomach Cancer, NQQLKWTPHIL (B08.01) Lynch syndrome NQQLKWTPHI (B08.01)QLKWTPHIL (B08.01) LMAN1 E305fs; +1 DDHDVLSFLTFQLTEP GPPRPPRAAC (B07.02)MSI + CRC, MSI  GKEPPTPDKEISEKEK PPRPPRAAC (B07.02) Uterine/EKYQEEFEHFQQELD Endometrium  KKKRGIPEGPPRPPRA Cancer, MSI  ACGGNI*Stomach Cancer, Lynch syndrome LMAN1 E305fs; −1 DDHDVLSFLTFQLTEPSLRRKYLRV (B08.01) MSI + CRC, MSI  GKEPPTPDKEISEKEK Uterine/EKYQEEFEHFQQELD Endometrium  KKKRNSRRATPTSKG Cancer, MSI  SLRRKYLRV*Stomach Cancer, Lynch syndrome MSH3 N385fs; +1 TKSTLIGEDVNPLIKLSAACHRRGCV (B08.01) MSI + CRC, MSI  DDAVNVDEIMTDTST Uterine/SYLLCISENKENVRDK Endometrium  KKGQHFYWHCGSAA Cancer, MSI  CHRRGCV*Stomach Cancer, Lynch syndrome MSH3 K383fs; −1 LYTKSTLIGEDVNPLIALWECSLPQA (A02.01) MSI + CRC, KLDDAVNVDEIMTDT CLIVSRTLL (B08.01)MSI + Uterine/ STSYLLCISENKENVR CLIVSRTLLL (A02.01, EndometriumDKKRATFLLALWECS B08.01) Cancer, MSI  LPQARLCLIVSRTLLL FLLALWECS (A02.01)Stomach Cancer, VQS* FLLALWECSL (A02.01, Lynch syndrome B08.01)IVSRTLLLV (A02.01) LIVSRTLLL (A02.01, B08.01) LIVSRTLLLV (A02.01)LLALWECSL (A02.01, B08.01) LPQARLCLI (B08.01, B07.02)LPQARLCLIV (B08.01) NVRDKKRATF (B08.01) SLPQARLCLI (A02.01, B08.01)NDUFC A70fs; +1 LPPPKLTDPRLLYIGFL FFCWILSCK (A03.01) MSI + CRC, 2GYCSGLIDNLIRRRPIA FFFCWILSCK (A03.01) MSI + Uterine/ TAGLHRQLLYITAFFFITAFFFCWI (A02.01) Endometrium CWILSCKT* LYITAFFFCW (A24.02)Cancer, MSI  YITAFFFCWI (A02.01) Stomach Cancer, Lynch syndrome NDUFCF69fs; −1 SLPPPKLTDPRLLYIGF ITAFFLLDI (A02.01) MSI + CRC, 2LGYCSGLIDNLIRRRPI LLYITAFFL (A02.01, MSI + Uterine/ ATAGLHRQLLYITAFFB08.01) Endometrium LLDIIL* LLYITAFFLL (A02.01, Cancer, MSI  A24.02)Stomach Cancer, LYITAFFLL (A24.02) Lynch syndrome LYITAFFLLD (A24.02)YITAFFLLDI (A02.01) RBM27 Q817; +1 NQSGGAGEDCQIFSTP GSNEVTTRY (A01.01)MSI + CRC, GHPKMIYSSSNLKTPS MPKDVNIQV (B07.02) MSI + Uterine/KLCSGSKSHDVQEVL TGSNEVTTRY (A01.01) Endometrium KKKTGSNEVTTRYEECancer, MSI  KKTGSVRKANRMPKD Stomach Cancer, VNIQVRKKQKHETRRLynch syndrome KSKYNEDFERAWRED LTIKR* RPL22 K16fs; +1 MAPVKKLVVKGGKKMSI + CRC, KEASSEVHS* MSI + Uterine/ Endometrium Cancer, MSI Stomach Cancer, Lynch syndrome RPL22 K15fs; −1 MAPVKKLVVKGGKK MSI + CRC,RSKF* MSI + Uterine/ Endometrium Cancer, MSI  Stomach Cancer,Lynch syndrome SEC31A I462fs; +1 MPSHQGAEQQQQQHH MSI + CRC,VFISQVVTEKEFLSR MSI + Uterine/ SDQLQQAVQSQGFIN Endometrium YCQKKN*Cancer, MSI  Stomach Cancer, Lynch syndrome SEC31A I462fs; −1MPSHQGAEQQQQQHH KKLMLLRLNL (A02.01) MSI + CRC, VFISQVVTEKEFLSRKLMLLRLNL (A02.01, MSI + Uterine/ SDQLQQAVQSQGFIN A03.01, B07.02,Endometrium YCQKKLMLLRLNLRK B08.01) Cancer, MSI  MCGPF*KLMLLRLNLR (A03.01) Stomach Cancer, LLRLNLRKM (B08.01) Lynch syndromeLMLLRLNL (B08.01) LMLLRLNLRK (A03.01) LNLRKMCGPF (B08.01)MLLRLNLRK (A03.01) MLLRLNLRKM (A02.01, A03.01, B08.01)NLRKMCGPF (B08.01) NYCQKKLMLL (A24.02) YCQKKLMLL (B08.01) SEC63K530fs; +1 AEVFEKEQSICAAEEQ FK.KK.TYTCAI (B08.01) MSI + CRC,PAEDGQGETNKNRTK 1TTVKATETK (A03.01) MSI + Uterine/ GGWQQKSKGPKKTAKSKKKETFK (A03.01) Endometrium KSKKKETFKKKTYTC KSKKKETFKK (A03.01)Cancer, MSI  AITTVKATETKAGKW KTYTCAITTV (A02.01, Stomach Cancer, SRWE*A24.02) Lynch syndrome TFKKKTYTC (B08.01) TYTCAITTV (A24.02)TYTCAITTVK (A03.01) YTCAITTVK (A03.01) SEC63 K529fs; −1 MAEVFEKEQSICAAEETAKSKKRNL (B08.01) MSI + CRC, QPAEDGQGETNKNRT MSI + Uterine/KGGWQQKSKGPKKT Endometrium AKSKKRNL* Cancer, MSI  Stomach Cancer,Lynch syndrome SLC35F C248fs; −1 NIMEIRQLPSSHALEA FALCGFWQI (A02.01)MSI + CRC, 5 KLSRMSYPVKEQESIL MSI + Uterine/ KTVGKLTATQVAKISFEndometrium FFALCGFWQICHIKKH Cancer, MSI  FQTHKLL* Stomach Cancer,Lynch syndrome SMAP1 KI72fs; +1 YEKKKYYDKNAIAIT MSI + CRC,NISSSDAPLQPLVSSPS MSI + Uterine/ LQAAVDKNKLEKEKE EndometriumKKKGREKERKGARKA Cancer, MSI  GKTTYS* Stomach Cancer, Lynch syndromeSMAP1 K171fs; −1 KYEKKKYYDKNAIAI LKKLRSPL (B08.01) MSI + CRC,TNISSSDAPLQPLVSSP SLKKVPAL (B08.01) MSI + Uterine/ SLQAAVDKNKLEKEKRKISNWSLKK (A03.01) Endometrium EKKRKRKREKRSQKS VPALKKLRSPL (B07.02)Cancer, MSI  RQNHLQLKSCRRKISN Stomach Cancer, WSLKKVPALKKLRSPLynch syndrome LWIF* TFAM E148fs; +1 IYQDAYRAEWQVYKE KRVNTAWKTK (A03.01)MSI + CRC, EISRFKEQLTPSQIMSL MTKKKRVNTA (B08.01) MSI + Uterine/EKEIMDKHLKRKAMT RVNTAWKTK (A03.01) Endometrium KKKRVNTAWKTKKTRVNTAWKTKK (A03.01) Cancer, MSI  SFSL* TKKKRVNTA (B08.01) lynch syndromeWKTKKTSFSL (B08.01) Lynch syndrome TFAM E148fs; −1 IYQDAYRAEWQVYKEMSI + CRC, EISRFKEQLTPSQIMSL MSI + Uterine/ EKEIMDKHLKRKAMT EndometriumKKKS* Cancer, MSI  Stomach Cancer, Lynch syndrome TGFBR2 P129fs; +1KPQEVCVAVWRKND MSI + CRC, ENITLETVCHDPKLPY MSI + Uterine/HDFILEDAASPKCIMK Endometrium EKKKAW* Cancer, MSI  Stomach Cancer,Lynch syndrome TGFBR2 K128fs: −1 EKPQEVCVAVWRKN ALMSAMTTS (A02.01)MSI + CRC, DENITLETVCHDPKLP AMTTSSSQK (A03.01, MSI + Uterine/YHDFILEDAASPKCIM A11.01) Endometrium KEKKSLVRLSSCVPVAAMTTSSSQKN (A03.01) Cancer, MSI  LMSAMTTSSSQKNITP CIMKEKKSL (B08.01)Stomach Cancer, AILTCC* CIMKEKKSLV (B08.01) Lynch syndromeIMKEKKSL (B08.01) IMK.EKK.SLV (B08.01) KSLVRLSSCV (A02.01)LVRLSSCVPV (A02.01) RLSSCVPVA (A02.01, A03.01) RLSSCVPVAL (A02.01)SAMTTSSSQK (A03.01, A11.01) SLVRLSSCV (A02.01) VPVALMSAM (B07.02)VRLSSCVPVA (A02.01) THAP5 K99fs; −1 VPSKYQFLCSDHFTPD KMRKKYAQK (A03.01)MSI + CRC, SLDIRWGIRYLKQTAV MSI + Uterine/ PTIFSLPEDNQGKDPS EndometriumKKNPRRKTWKMRKK Cancer, MSI  YAQKPSQKNHLY* Stomach Cancer, Lynch syndromeTTK R854fs; −1 GTTEEMKYVLGQLVG FVMSDTTYK (A03.01) MSI + CRC,LNSPNSILKAAKTLYE FVMSDTTYKI (A02.01) MSI + Uterine/ HYSGGESHNSSSSKTFKTFEKKGEK (A03.01) Endometrium EKKGEKNDLQLFVMS LFVMSDTTYK (A03.01)Cancer, MSI  DTTYKIYWTVILLNPC MSDTTYKIY (AO 1.01) Stomach Cancer,GNLHLKTTSL* VMSDTTYKI (A02.01) Lynch syndrome VMSDTTYKIY (A01.01) XPOTF126fs; −1 QQLIRETLISWLQAQM YLTKWPKFFL (A02.01) MSI + CRC,LNPQPEKTFIRNKAAQ MSI + Uterine/ VFALLFVTEYLTKWP Endometrium KFFLTFSQ*Cancer, MSI  Stomach Cancer, Lynch syndrome TABLE 1C FRAMESHIFT ¹ APCV1352fs AKFQQCHSTLEPNPA FLQERNLPP (A02.01) CRC, LUAD, F1354fsDCRVLVYLQNQPGTK FRRPHSCLA (B08.01) UCEC, STAD Q1378fs LLNFLQERNLPPKVVLLIVLRVVRL (B08.01) S1398fs RHPKVHLNTMFRRPH LLSVHLIVL (A02.01,SCLADVLLSVHLIVLR B08.01) VVRLPAPFRVNHAVE W* APC S1421fs APVIFQIALDKPCHQAEVKHLHHLL (B08.01) CRC, LUAD, R1435fs EVKHLHHLLKQLKPSHLHHLLKQLK (A03.01) UCEC, STAD T1438fs EKYLKIKHLLLKRERVHLLLKRERV (B08.01) P1442fs DLSKLQ* KIKHLLLKR (A03.01) P1443fsKPSEKYLKI (B07.02) V1452fs KYLK.1KHLL (A24.02) P1453fsKYLKIKHLLL (A24.02) K1462fs LLKQLKPSEK (A03.01) E1464fsLLKRERVDL (B08.01) LLLKRERVDL (B08.01) QLKPSEKYLK (A03.01)YLKIKHLLL (A02.01, B08.01) YLKIKF1LLLK (A03.01) APC T1487fsMLQFRGSRFFQMLILY ILPRKVLQM (B08.01) CRC, LUAD, H1490fs YILPRKVLQMDFLVHPKVLQMDFLV (A02.01, UCEC, STAD L1488fs A* A24.02) LPRKVLQMDF (B07.02,B08.01) LQMDFLVHPA (A02.01) QMDFLVHPA (A02.01) YILPRKVLQM (A02.01,B08.01) ARID1A Q1306fs ALGPHSRISCLPTQTR APSPASRLQC (B07.02) STAD, UCEC,S1316fs GCILLAATPRSSSSSSS HPLAPMPSKT (B07.02) BLCA, BRCA, Y1324fsNDMIPMAISSPPKAPL ILPLPQLLL (A02.01) LUSC, CESC, T1348fs LAAPSPASRLQCINSNLLLSADQQA (A02.01) KIRC, UCS G135lfs SRITSGQWMAHMALL LPTQTRGCI (B07.02)G1378fs PSGTKGRCTACHTAL LPTQTRGCIL (B07.02) P1467fs GRGSLSSSSCPQPSPSLRISCLPTQTR (A03.01) PASNKLPSLPLSKMYT SLAETVSLH (A03.01) TSMAMPILPLPQLLLSTPRSSSSSS (B07.02) ADQQAAPRTNFHSSE TPRSSSSSSS (B07.02) AETVSLHPLAPMPSKTCHHK* ARID1A S674fs AHQGFPAAKESRVIQL ALPPVLLSL (A02.01) STAD, UCEC,P725fs SLLSLLIPPLTCLASEA ALPPVLLSLA (A02.01) BLCA, BRCA, R727fsLPRPLLALPPVLLSLA ALPRPLLAL (A02.01) LUSC, CESC, I736fs QDHSRLLQCQATRCHASRTASCIL (B07.02) KIRC, UCS LGHPVASRTASCILP* EALPRPLLAL (B08.01)HLGHPVASR (A03.01) HPVASRTAS (B07.02) HPVASRTASC (B07.02)IIQLSLLSLL (A02.01) IQLSLLSLL (A02.01) IQLSLLSLLI (A02.01, A24.02)LLALPPVLL (A02.01) LLIPPLTCL (A02.01) LLIPPLTCLA (A02.01)LLSLLIPPL (A02.01) LLSLLIPPLT (A02.01) LPRPLLALPP (B07.02)QLSLLSLLI (A02.01) RLLQCQATR (A03.01) RPLLALPPV (B07.02)RPLLALPPVL (B07.02) SLAQDHSRL (A02.01) SLAQDHSRLL (A02.01)SLLIPPLTCL (A02.01) SLLSLLIPP (A02.01) SLLSLLIPPL (A02.01, B08.01)ARID1A G414fs PILAATGTSVRTAART AAATSAASTL (B07.02) STAD, UCEC, Q473fsWVPRAAIRVPDPAAV AAIPASTSAV (B07.02) BLCA, BRCA, H477fs PDDHAGPGAECHGRPAIPASTSAV (A02.01) LUSC, CESC, S499fs LLYTADSSLWTTRPQALPAGCVSSA (A02.01) KIRC, UCS P504fs RVWSTGPDSILQPAKSAPLLTATGSV (B07.02) Q548fs SPSAAAATLLPATTVP APVLSASIL (B07.02) P549fsDPSCPTFVSAAATVST ATLLPATTV (A02.01) TTAPVLSASILPAAIPAATVSTTTAPV (A02.01) STSAVPGSIPLPAVDD AVPANCLFPA (A02.01)TAAPPEPAPLLTATGS CLFPAALPST (A02.01) VSLPAAATSAASTLDA CPTFVSAAA (B07.02)LPAGCVSSAPVSAVPA FPAALPSTA (B07.02) NCLFPAALPSTAGAIS FPAALPSTAG (B07.02)RFIWVSGILSPLNDLQ* GAECHGRPL (B07.02) GAISRF1WV (A02.01)ILPAAIPAST (A02.01) IWVSGILSPL (A24.02) LLTATGSVSL (A02.01)LLYTADSSL (A02.01) LPAAATSAA (B07.02) LPAAATSAAS (B07.02)LPAAIPAST (B07.02) LPAGCVSSA (B07.02) LPAGCVSSAP (B07.02)LYTADSSLW (A24.02) QPAKSSPSA (B07.02) QPAKSSPSAA (B07.02)RFIWVSGIL (A24.02) RPQRVWSTG (B07.02) RVWSTGPDSI (A02.01)SAVPGSIPL (B07.02) SILPAAIPA (A02.01) SLPAAATSA (A02.01)SLPAAATSAA (A02.01) SLWTTRPQR (A03.01) SLWTTRPQRV (A02.01)SPSAAAATL (B07.02) SPSAAAATLL (B07.02) TLDALPAGCV (A02.01)TVSTTTAPV (A02.01) VLSASILPA (A02.01) VLSASILPAA (A02.01)VPANCLFPA (B07.02) VPANCLFPAA (B07.02) VPDPSCPTF (B07.02)VPGSIPLPA (B07.02) VPGSIPLPAV (B07.02) WVSGILSPL (A02.01)YTADSSLWTT (A02.01) ARID1A T433fs PCRAGRRVPWAASLI APAGMVNRA (B07.02)STAD, UCEC, A441fs HSRFLLMDNKAPAGM ASLHRRSYL (B08.01) BLCA, BRCA, Y447fsVNRARLHITTSKVLTL ASLHRRSYLK (A03.01) LUSC, CESC, P483fs SSSSHPTPSNHRPRPLFLLMDNKAPA (A02.01) KIRC, UCS P484fs MPNLR1SSSHSLNHHS HPRRSPSRL (B07.02,P504fs SSPLSLHTPSSHPSLHI B08.01) S519fs SSPRLHTPPSSRRHSSTHPSLHISSP (B07.02) H544fs PRASPPTHSHRLSLLTS HRRSYLKIHL (B08.01) P549fsSSNLSSQHPRRSPSRLR HSRFLLMDNK (A03.01) P554fs ILSPSLSSPSKLPIPSSKLPIPSSASL (A02.01) Q363fs ASLHRRSYLKIFILGLR KVLTLSSSSFI (A03.01)HPQPPQ* LIHSRFLLM (B08.01) LLMDNKAPA (A02.01) LMDNKAPAGM (A02.01)LPIPSSASL (B07.02) MPNLRISSS (B07.02,  B08.01) MPNLRISSSH (B07.02)NLRISSSHSL (B07.02, B08.01) PPTHSHRLSL (B07.02) RAGRRVPWAA (B08.01)RARLHITTSK (A03.01) RISSSHSLNH (A03.01) RLHTPPSSR (A03.01)RLHTPPSSRR (A03.01) RLRILSPSL (A02.01, B07.02, B08.01)RPLMPNLRI (B07.02) RPRPLMPNL (B07.02) SASLHRRSYL (B07.02, B08.01)SLHISSPRL (A02.01) SLHRRSYLK (A03.01) SLHRRSYLKI (B08.01)SLIHSRFLL (A02.01) SLIHSRFLLM (A02.01, B08.01) SLLTSSSNL (A02.01)SLNHHSSSPL (A02.01, B07.02, B08.01) SLSSPSKLP1 (A02.01)SPLSLHTPS (B07.02) SPLSLHTPSS (B07.02) SPPTHSHRL (B07.02)SPRLHTPPS (B07.02) SPRLHTPPSS (B07.02) SPSLSSPSKL (B07.02)SYLK1HLGL (A24.02) TPSNHRPRPL (B07.02, B08.01) TPSSHPSLHI (B07.02)ARID1A A2l37fs RTNPTVRMRPHCVPFW CVPFWTGRIL (B07.02) STAD, UCEC, P2139fsTGRILLPSAASVCPIP HCVPFWTGRIL (B07.02) BLCA, BRCA, L1970fsFEACHLCQAMTLRCP ILLPSAASV (A02.01) LUSC, CESC, V1994fs NTQGCCSSWAS*ILLPSAASVC (A02.01) KIRC, UCS LLPSAASVCPI (A02.01) LPSAASVCPI (B07.02)MRPHCVPF (B08.01) RILLPSAASV (A02.01) RMRPHCVPF (A24.02, B07.02, B08.01)RMRPHCVPFW (A24.02) RTNPTVRMR (A03.01) SVCPIPFEA (A02.01)TVRMRPHCV (B08.01) TVRMRPHCVPF (B08.01) VPFWTGRIL (B07.02)VPFWTGRILL (B07.02) VRMRPHCVPF (B08.01) ARID1A N756fs TNQALPKIEVICRGTPAMVPRGVSM (B07.02, STAD, UCEC, S764fs RCPSTVPPSPAQPYLR B08.01)BLCA, BRCA, T783fs VSLPEDRYTQAWAPT AMVPRGVSM A (A02.01) LUSC, CESC,Q799fs SRTPWGAMVPRGVS AWAPTSRTPW (A24.02) KIRC, UCS A817fsMAHKVATPGSQTIMP CPMPTTPVQA (B07.02) CPMPTTPVQAWLEA* CPSTVPPSPA (B07.02)GAMVPRGVSM (B07.02, B08.01) MPCPMPTTPV (B07.02) MPTTPVQAW (B07.02)MPTTPVQAWL (B07.02) SLPEDRYTQA (A02.01) SPAQPYLRV (B07.02)SPAQPYLRVS (B07.02) TIMPCPMPT (A02.01) TPVQAWLEA (B07.02)TSRTPWGAM (B07.02) VPPSPAQPYL (B07.02) VPRGVSMAH (B07.02) β2M N62fsRMERELKKWSIQTCL CLSARTGLSI (B08.01) CRC, STAD, E67fs SARTGLSISCTTLNSPPCTTLNSPPLK (A03.01) SKCM, HNSC L74fs LKKMSMPAV* GLSISCTTL (A02.01) F82fsSPPLKKMSM (B07.02, T91fs B08.01) E94fs TLNSPPLKK (A03.01)TTLNSPPLK (A03.01) TTLNSPPLKK (A03.01) β2M L13fs LCSRYSLFLAWRLSSVLQRFRFTHV (B08.01) CRC, STAD, S14fs LQRFRFTHVIQQRMES LQRFRFTHVI (B08.01)SKCM, HNSC QIS* RLSSVLQRF (A24.02) RLSSVLQRFR (A03.01)VLQRFRFTHV (A02.01, B08.01) CDH1 A691fs RSACVTVKGPLASVGASVGRHSLSK (A03.01) ILC LumA Breast P708fs RHSLSKQDCKFLPFWKFLPFWGFL (A24.02) Cancer L711fs GFLEEFLLC* LASVGRHSL (B07.02)LPFWGFLEEF (B07.02) PFWGFLEEF (A24.02) SVGRHSLSK (A03.01) CDH1 H121fsIQWGTTTAPRPIRPPFL APRPIRPPF (B07.02) ILC LumA Breast P126fsESKQNCSHFPTPLLAS APRPIRPPFL (B07.02) Cancer HI28fs EDRRETGLFLPSAAQKAQKMKKAFIFL (B08.01) N144fs MKKAHFLKTWFRSNP FLPSAAQKM (A02.01) V157fsTKTKKARFSTASLAKE GLFLPSAAQK (A03.01) PI59fs LTHPLLVSLLLKEKQDHPLLVSLLL (B07.02) N166fs G* KAHFLKTWFR (A03.01) N181fsKARFSTASL (B07.02) F189fs KMKKAHFLK (A03.01) P201fs KTWFRSNPTK (A03.01)F205fs LAKELTHPL (B07.02, B08.01) LAKELTHPLL (B08.01)NPTKTKKARF (B07.02) QKMKKAHFL (B08.01) RFSTASLAK (A03.01)RPIRPPFLES (B07.02) RSNPTKTKK (A03.01) SLAKELTHPL (A02.01, B08.01)TKKARFSTA (B08.01) CDH1 V114fs PTDPFLGLRLGLHLQK GLRFWNPSR (A03.01)ILC LumA Breast P127fs VFHQSHAEYSGAPPPP ISQLLSWPQK (A03.01) CancerV132fs PAPSGLRFWNPSRIAH RIAHISQLL (A02.01) P160fs ISQLLSWPQKTEERLGRLGYSSHQL (A02.01) YSSHQLPRK* SQLLSWPQK (A03.01) SRIAHISQL (B08.01)WPQKTEERL (B07.02) YSSHQLPRK (A03.01) CDH1 L731fs FCCSCCFFGGERWSKSCPQRMTPGTT (B07.02) ILC LumA Breast R749fs PYCPQRMTPGTTFITMEAEKRTRTL (B08.01) Cancer E757fs MKKEAEKRTRTLT* GTTFITMMK (A03.01)G759fs GTTFITMMKK (A03.01) ITMMKKEAEK (A03.01) RMTPGTTFI (A02.01)SPYCPQRMT (B07.02) TMMKKEAEK (A03.01) TPGTTFITM (B07.02)TPGTTFITMM (B07.02) TTFITMMKK (A03.01) CDH1 S19fs WRRNCKAPVSLRKSVCPGATWREA (B07.02) ILC LumA Breast E24fs QTPARSSPARPDRTRRCPGATWREAA (B07.02) Cancer S36fs LPSLGVPGQPWALGA RSRCPGATWR (A03.01)AASRRCCCCCRSPLGS TPRATRSRC (B07.02) ARSRSPATLALTPRAT RSRCPGATWREAASW AE*GATA3 P394fs PGRPLQTHVLPEPHLA HVLPEPHLAL (B07.02) Breast Cancer P387fsLQPLQPHADHAHADA RPLQTHVLPE (B07.02) S398fs PAIQPVLWTTPPLQHGVLWTTPPLQH (A03.01) H400fs HRHGLEPCSMLTGPP M401fs ARVPAVPFDLHFCRSSS408fs IMKPKRDGYMFLKAE P409fs SKIMFATLQRSSLWCL S408fs CSNH* P409fsT419fs H424fs P425fs S427fs F431fs S430fs H434fs H435fs S438fs M443fsG444fs *445fs GATA3 P426fs PRPRRCTRHPACPLDH APSESPCSPF (B07.02)Breast Cancer H434fs TTPPAWSPPWVRALL CPLDHTTPPA (B07.02) P433fsDAHRAPSESPCSPFRL FLQEQYHEA (A02.01, T441fs AFLQEQYHEA* B08.01)RLAFLQEQYH (A03.01) SPCSPFRLAF (B07.02) SPPWVRALL (B07.02)YPACPLDHTT (B07.02) MLL2 P519fs TRRCHCCPHLRSHPCP ALHLRSCPC (B08.01)STAD, BLCA, E524fs HHLRNHPRPHHLRHH CLHHRRFILV (B08.01) CRC, HNSC, P647fsACHHHLRNCPHPHFL CLHHRRHLVC (B08.01) BRCA S654fs RHCTCPGRWRNRPSLCLHRKSHPHL (B08.01) L656fs RRLRSLLCLPHLNHHL CLRSHACPP (B08.01) R755fsFLHWRSRPCLHRKSH CLRSHTCPP (B08.01) L761fs PHLLHLRRLYPHHLKCLWCHACLFI (A03.01) Q773fs HRPCPHHLKNLLCPR CPHHLK.NHL (BO7.02)HLRNCPLPRHLKHLA CPHHLKNLL (B07.02) CLHHLRSHPCPLHLKS CPHHLRTRL (B07.02,HPCLHHRRHLVCSHH B08.01) LKSLLCPLHLRSLPFP CPLHLRSLPF (B07.02,HHLRHHACPHHLRTR B08.01) LCPHHLKNHLCPPHLR CPLPRHLKHL (B07.02,YRAYPPCLWCHACLH B08.01) RLRNLPCPHRLRSLPR CPLSLRSHPC (B07.02)PLHLRLHASPHHLRTP CPRHLRNCPL (B07.02, PHPHHLRTHLLPHHRR B08.01)TRSCPCRWRSHPCCH FPHHLRHHA (B07.02, YLRSRNSAPGPRGRTC B08.01)HPGLRSRTCPPGLRSH FPHHLRHHAC (B07.02, TYLRRLRSHTCPPSLR B08.01)SHAYALCLRSHTCPPR GLRSRTCPP (B08.01) LRDHICPLSLRNCTCP HACLHRLRNL (B08.01)PRLRSRTCLLCLRSHA HLACLHHLR (A03.01) CPPNLRNHTCPPSLRS HLCPPHLRY (A03.01)HACPPGLRNRICPLSL HLCPPHLRYR (A03.01) RSHPCPLGLKSPLRSQ HLKHLACLH (A03.01)ANALHLRSCPCSLPLG HLKHRPCPH (B08.01) NHPYLPCLESQPCLSL HLKNHLCPP (B08.01)GNHLCPLCPRSCRCPH HLKSHPCLH (A03.01) LGSHPCRLS* HLKSLLCPL (A02.01,B08.01) HLLHLRRLY (A03.01) HLRNCPLPR (A03.01) HLRNCPLPRH (A03.01)HLRRLYPHHL (B08.01) HLRSHPCPL (B07.02, B08.01) HLRSHPCPLH (A03.01)HLRSLPFPH (A03.01) HLRTRLCPH (A03.01, B08.01) HLVCSHHLK (A03.01)HPCLHHRRHL (B07.02, B08.01) HPGLRSRTC (B07.02) HPHLLHLRRL (B07.02,B08.01) HRKSHPHLL (B08.01) HRRTRSCPC (B08.01) KSHPHLLHLR (A03.01)KSLLCPLHLR (A03.01) LLCPLHLRSL (A02.01, B08.01) LLHLRRLYPH (B08.01)LPRHLKHLA (B07.02) LPRHLKHLAC (B07.02, B08.01) LRRLRSHTC(B08.01) PTEN1122fs SWKGTNWCNDMCIFI FITSGQIFK (A03.01) UCEC, PRAD, 1135fsTSGQIFKGTRGPRFLW IFITSGQIF (A24.02) SKCM, STAD, A148fs GSKDQRQKGSNYSQSSQSEALCVL (A02.01) BRCA, LUSC, L152fs EALCVLL* SQSEALCVLL (A02.01)KIRC, LIHC, D162fs KIRP, GBM I168fs PTEN L265fs KRTKCFTFG* UCEC, PRAD,K266fs SKCM, STAD, BRCA, LUSC, KIRC, LIHC, KIRP, GBM PTEN A39fsPIFIQTLLLWDFLQKD AYTGTILMM (A24.02) UCEC, PRAD, E40fs LKAYTGTILMM*DLKAYTGTIL (B08.01) SKCM, STAD, V45fs BRCA, LUSC, R47fs KIRC, LIHC,N48fs KIRP, GBM PTEN T319fs QKMILTKQIKTKPTDT ILTKQIKTK (A03.01)UCEC, PRAD, T321fs FLQILR* KMILTKQIK (A03.01) SKCM, STAD, K327fsKPTDTFLQI (B07.02) BRCA, LUSC, A328fs KPTDTFLQIL (B07.02) KIRC, LIHC,A333fs MILTKQIKTK (A03.01) KIRP, GBM PTEN N63fs GFWIQSIKTITRYTIITRYTIFVLK (A03.01) UCEC, PRAD, E73fs FVLKDIMTPPNLIAE LIAELHNIL (A02.01)SKCM, STAD, A86fs LHNILLKTITHHS* LIAELHNILL (A02.01) BRCA, LUSC, N94fsMTPPNLIAEL (A02.01) KIRC, LIHC, NLIAELHNI (A02.01) KIRP, GBMNLIAELHNIL (A02.01) RYTIFVLKDI (A24.02) TITRYTIFVL (A02.01)TPPNLIAEL (B07.02) PTEN T202fs NYSNVQWRNLQSSVC FLQFRTHTT (A02.01,UCEC, PRAD, G209fs GLPAKGEDIFLQFRTH B08.01) SKCM, STAD, C211fsTTGRQVHVL* LPAKGEDIFL (B07.02) BRCA, LUSC, I224fs LQFRTHTTGR (A03.01)KIRC, LIHC, G230fs NLQSSVCGL (A02.01) KIRP, GBM P231fsSSVC'GLPAK (A03.01) R233fs VQWRNLQSSV (A02.01) D236fs PTEN G251fsYQSRVLPQTEQDAKK GQNVSLLGK (A03.01) UCEC, PRAD, E256fs GQNVSLLGKYILHTRTHTRTRGNLRK (A03.01) SKCM, STAD, K260fs RGNLRKSRKWKSM*ILHTRTRGNL (B08.01) BRCA, LUSC, Q261fs KGQNVSLLGK (A03.01) KIRC, LIHC,L265fs LLGKYILHT (A02.01) KIRP, GBM M270fs LRKSRKWKSM (B08.01) H272fsSLLGKYILH (A03.01) T286fs SLLGKYILHT (A02.01) E288fs TP53 A70fsSSQNARGCSPRGPCTS CTSPLLAPV (A02.01) BRCA, CRC, P72fs SSYTGGPCTSPLLAPVIFPENLPGQL (B07.02) LUAD, PRAD, A76fs FCPFPENLPGQLRFPSGLLAFWDSQV (A02.01) HNSC, LUSC, A79fs GLLAFWDSQVCDLHV IFCPFPENL (A24.02)PAAD, STAD, P89fs LPCPQQDVLPTGQDLP LLAFWDSQV (A02.01) BLCA, OV, LIHC,W91fs CAAVG* LLAPVIFCP (A02.01) SKCM, UCEC, S96fs LLAPVIFCPF (A02.01,LAML, UCS, V97fs A24.02) KICH, GBM, ACC V97fs LPCPQQDVL (B07.02) G108fsRFPSGLLAF (A24.02) G117fS RFPSGLLAFW (A24.02) S121fs SPLLAPVIF (B07.02)V122fs SPRGPCTSS (B07.02) C124fs SPRGPCTSSS (B07.02) K139fsSQVCDLHVL (A02.01) V143fs VIFCPFPENL (A02.01) TP53 V173fs GAAPTMSAAQIAMVAMVWPLLSI (A02.01) BRCA, CRC, H178fs WPLLSILSEWKEICVWAMVWPLLS1L (A02.01) LUAD, PRAD, D186fs SIWMTETLFDIVWWCAQIAMVWPL (A02.01, HNSC, LUSC, H193fs PMSRLRLALTVPPSTT A24.02)PAAD, STAD, L194fs TTCVTVPAWAA* AQIAMVWPLL (A02.01) BLCA, OV, LIHC,E198fs CPMSRLRLA (B07.02, SKCM, UCEC, V203fs B08.01) LAML, UCS, E204fsCPMSRLRLAL (B07.02, KICH, GBM, ACC L206fs B08.01) D207fsIAMVWPLLSI (A02.01, N210fs A24.02, B08.01) T211fs ILSEWKEICV (A02.01)F212fs IVWWCPMSR (A03.01) V225fs IVWWCPMSRL (A02.01) S241fsIWMTETLFDI (A24.02) LLSILSEWK (A03.01) MSAAQIAMV (A02.01)MSRLRLALT (B08.01) MSRLRLALTV (B08.01) MVWPLLS1L (A02.01)RLALTVPPST (A02.01) TLFDIVWWC (A02.01) TLFD1VWWCP (A02.01)TMSAAQIAMV (A02.01) VWSIWMTETL (A24.02) WMTETLFD1 (A02.01, A24.02)WMTETLFD1V (A01.01, A02.01) TP53 R248fs TGGPSSPSSHWKTPVVALRCVFVPV (A02.01, BRCA, CRC P250fs IYWDGTALRCVFVPV B08.01) LUAD, PRAD,S260fs LGETGAQRKRISARK ALRCVFVPVL (A02.01, HNSC, LUSC, N263fsGSLTTSCPQGALSEHC B08.01) PAAD, STAD, G266fs PYFPAPLPSQRRNHWALSEHCPTT (A02.01) BLCA, OV, LIHC, N268fs MENISPFRSVGVSASRAQRKRISARK (A03.01) SKCM, UCEC, V272fs CSES* GAQRKRISA (B08.01)LAML, UCS, V274fs HWMENISPF (A24.02) KICH, GBM, ACC P278fsLPSQRRNHW (B07.02) D281fs LPSQRRNHWM (B07.02, R282fs B08.01) T284fsNISPFRSVGV (A02.01) E285fs RISARKGSL (B07.02, L289fs B08.01) K292fsSPFRSVGVSA (B07.02) P301fs SPSSHWKTPV (B07.02, S303fs B08.01) T312fsTALRCVFVPV (A02.01) S314fs VIYWDGTAL (A02.01) K319fs VIYWDGTALR (A03.01)K320fs VLGETGAQRK (A03.01) P322fs Y327fs F328fs L330fs R333fs R335fsR337fs E339fs TP53 S149fs FHTPARHPRPRHGHL HPRPRHGHL (B07.02, BRCA, CRC,P151fs QAVTAHDGGCEALPP B08.01) LUAD, PRAD, P152fs P* HPRPRHGHLQ (B07.02)HNSC, LUSC, V157fs RPRHGHLQA (B07.02) PAAD, STAD, Q165fsRPRHGHLQAV (B07.02, BLCA, OV, LIHC, S166fs B08.01) SKCM, UCEC, H168fsLAML, UCS, V173fs KICH, GBM, ACC TP53 P47fs CCPRTILNNGSLKTQVGSLKTQVQMK (A03.01) BRCA, CRC, D48fs QMKLPECQRLLPPWPLPPGPCHLLSL (B07.02) LUAD, PRAD, D49fs HQQLLIIRRPLIIQPPPRTILNNGSLK (A03.01) HNSC, LUSC, Q52fs GPCHLLSLPRKPTRAASLKTQVQMK (A03.01) PAAD, STAD, F54fs TVSVWASCILGQPSL*SLKTQVQMKL (B08.01) BLCA, OV, LIHC, E56fs TILNNGSLK (A03.01) SKCM, UCEC,P58fs LAML, UCS, P60fs KICH, GBM, ACC E62fs M66fs P72fs V73fs P75fsA78fs P82fs P85fs S96fs P98fs T102fs Y103fs G108fs F109fs R110fs G117fsTP53 L26fs VRKHFQTYGNYFLKT CPPCRPKQWM (B07.02) BRCA, CRC, P27fsTFCPPCRPKQWMI* TTFCPPCRPK (A03.01) LUAD, PRAD, P34fs HNSC, LUSC, P36fsPAAD, STAD, A39fs BLCA, OV, LIHC, Q38fs SKCM, UCEC, LAML, UCS,KICH, GBM, ACC TP53 C124fs LARTPLPSTRCFANWP CFANWPRPAL (A24.02)BRCA, CRC, L130fs RPALCSCGLIPHPRPA FANWPRPAL (B07.02, LUAD, PRAD, N131fsAPSAPWPSTSSHST* B08.01) HNSC, LUSC, C135fs GLIPHPRPA (A02.01)PAAD, STAD, K139fs HPRPAPASA (B07.02, BLCA, OV, LIHC, A138fs B08.01)SKCM, UCEC, T140fs HPRPAPASAP (B07.02) LAML, UCS, V143fsIPHPRPAPA (B07.02, KICH, GBM, ACC Q144fs B08.01) V147fsIPHPRPAPAS (B07.02) T150fs RPALCSCGL (B07.02) P151fs RPALCSCGLI (B07.02)P152fs TPLPSTRCF (B07.02) G154fs WPRPALCSC (B07.02) R156fsWPRPALCSCG (B07.02) R158fs A161fs VHL L178fs ELQETGHRQVALRRSALRRSGRPPK (A03.01) KIRC, KIRP D179fs GRPPKCAERPGAADT GLVPSLVSK (A03.01)L184fs GAHCTSTDGRLKISVE KISVETYTV (A02.01) T202fs TYTVSSQLLMVLMSLLLMVLMSLDL (A02.01, R205fs DLDTGLVPSLVSKCLI B08.01) D2I3fs LRVK*LMSLDLDTGL (A02.01) G212fs LMVLMSLDL (A02.01) LVSKCLILRV (A02.01)QLLMVLMSL (A02.01, B08.01) RPGAADTGA (B07.02) RPGAADTGAH (B07.02)SLDLDTGLV (A02.01) SLVSKCLIL (A02.01, B08.01) SQLLMVLMSL (A02.01)TVSSQLLMV (A02.01) TYTVSSQLL (A24.02) TYTVSSQLLM (A24.02)VLMSLDLDT (A02.01) VPSLVSKCL (B07.02) VSKCL1LRVK (A03.01)YTVSSQLLM (A01.01) YTVSSQLLMV (A02.01) VHL L158fs KSDASRLSGA* KIRC,KIRPK159fs R161fs Q164fs VHL P146fs RTAYFCQYHTASVYS FCQYHTASV (B08.01)KIRC, KIRP I147fs ERAMPPGCPEPSQA* F148fs L158fs VHL S68fsTRASPPRSSSAIAVRAS CPYGSTSTA (B07.02) KIRC, KIRP S72fs CCPYGSTSTASRSPTQCPYGSTSTAS (B07.02) I75fs RCRLARAAASTATEV LARAAASTAT (B07.02) S80fsTFGSSEMQGHTMGFW MLTDSLFLP (A02.01) P86fs LTKLNYLCHLSMLTDPPRSSSAIAV (B07.02) P97fs SLFLPISHCQCIL* RAAASTATEV (B07.02) 1109fsSPPRSSSAI (B07.02) H115fs SPPRSSSAIA (B07.02) L116fs SPTQRCRLA (B07.02)G123fs TQRCRLARA (B08.01) T124fs TQRCRLARAA (B08.01) N131fs L135fsV137fs G144fs D143fs I147fs VHL K171fs SSLRITGDWTSSGRSTKIWKTTQMCR (A03.01) KIRC, KIRP P172fs KIWKTTQMCRKTWSG*WTSSGRSTK (A03.01) N174fs L178fs D179fs L188fs VHL V62fs RRRRGGVGRRGVRPGALGELARAL (A02.01) KIRC, KIRP V66fs RVRPGGTGRRGGDGG AQLRRRAAA (B08.01)Q73fs RAAAARAALGELARA AQLRRRAAAL (B08.01) V84fs LPGHLLQSQSARRAAARRAARMAQL (B08.01) F91fs RMAQLRRRAAALPNA HPQLPRSPL (B07.02, T100fsAAWHGPPHPQLPRSP B08.01) P103fs LALQRCRDTRWASG* HPQLPRSPLA (B07.02)S111fs LARALPGHL (B07.02) L116fs LARALPGHLL (B07.02) H115fsMAQLRRRAA (B07.02, D126fs B08.01) MAQLRRRAAA (B07.02, B08.01)QLRRRAAAL (B07.02, B08.01) RAAALPNAAA (B07.02) RMAQLRRRAA (B07.02,B08.01) SQSARRAARM (B08.01) TABLE 1D CRYPTIC EXON ¹ AR-v7 cryptic SCKVFFKRAAEGKQK GMTLGEKFRV (A02.01) Prostate Cancer, finalYLCASRNDCTIDKFRR RVGNCKHLK (A03.01) Castration-resistant exonKNCPSCRLRKCYEAG Prostate Cancer MTLGEKFRVGNCKHL KMTRP*TABLE 1E OUT OF FRAME FUSIONS ^(1,3) AC0119 AC011997.1: MAGAPPPASLPPCSLIGPSEPGNN1 (B07.02) LUSC, Breast 97.1:LR LRRC69 SDCCASNQRDSVGVGPK1CNESASRK (A03.01) Cancer, Head  RC69 *out-of- SEP:G: NNIKICNESA andframe SRK* Neck Cancer, LUAD EEF1DP EEFIDP3:FR HGWRPFLPVRARSRWGIQVLNVSLK (A03.01) Breast Cancer 3 Y *out-of- NRRLDVTVANGR:S: WIQVLNVSLK (A03.01) frame KYGWSLLRVPQVNG KSSSNVISY (A01.01,IQVLNVSLKSSSNVI A03.01) SYE* KYGWSLLRV (A24.02) RSWKYGWSL (A02.01)SLKSSSNVI (B08.01) SWKYGWSLL (A24.02) TVANGRSWK (A03.01)VPQVNGIQV (B07.02) VPQVNGIQVL (B07.02) VTVANGRSWK (A03.01)WSLLRVPQV (B08.01) MADIL MADIL1:MA RLKEVFQTKIQEFRKA HPGDCLIFKL (B07.02)CLL 1:MAFK FK CYTLTGYQIDITTENQ KLRVPGSSV (B07.02) YRLTSLYAEHPGDCLIKLRVPGSSVL (B07.02) FK:: LRVPGSSVLVTV RVPGSSVLV (A02.01) PGL*SVLVTVPGL (A02.01) VPGSSVLVTV (B07.02) PPP1R1 PPP1R1BST AEVLKVIRQSAGQKTALLLRPRPPR (A03.01) Breast Cancer B:STAR ARD3 TCGQGLEGPWERPPPLALSALLLRPR (A03.01) D3 DESERDGGSEDQVEDP ALS:A: LLLRPRPPRP EVGAHQDEQAAQGADPRLGAQPACRGLP GLLTVPQPEPLLAPP SAA*Table 1F IN FRAME DELETIONS and FUSIONS ^(1,2) BCR:AB BCRABLERAEWRENIREQQKK LTINKEEAL  CML, AML L CFRSFSLTSVELQMLT (A02.01, B08.01)NSCVKLQTVHSIPLTI NKE::EALQRPVASDF EPQGLSEAARWNSK ENLLAGPSENDPNLFVALYDFVASG BCR:AB BCRABL ELQMLTNSCVKLQTVH IVHSATGFK (A03.01) CML, AML LSIPLTINKEDDESPGL ATGFKQSSK (A03.01) YGFLNVIVHSATGFKQ SS:K:ALQRPVASDFEPQGLSEAARWNSKE NLLAGPSENDPNLFV ALYDFVASGD C11orf9 Cllorf95:ISNSWDAHLGLGACG ELFPLIFPA Supretentorial 5:RELA RELA EAEGLGVQGAEEEEEE(A02.01, B08.01) ependyomas EEEEEEEGAGVPACPP KGPELFPLI KGP:E:LFPLIFPAEP(A02.01, A24.02) AQASGPYVEIIEQPK KGPELFPLIF (A24.02) QRGMRFRYKCEGRSAGSIPGERSTD CBFBM: (variant LQRLDGMGCLEFDEE AML YH11 “type a”)RAQQEDALAQQAFEE ARRRTREFEDRDRSH REEME::VHELEKSKR ALETQMEEMKTQLEELEDELQATEDAKL RLEVNMQALKGQF CD74R (exon6: KGSFPENLRHLKNTMKPTDAPPKAGV (B07.02) NSCLC,  OS1 exon32) ETIDWKVFESWMHH CrizotinibWLLFEMSRHSLEQKP resistance TDAPPK::AGVPNKPG IPKLLEGSKNSIQVVEKAEDNGCRITYYILEI RKSTSNNLQNQ EGFR EGFRvIII MRPSGTAGAALLALLALEEKKGNYV (A02.01) GBM (internal AALCPASRALEEKK:G deletion):NYVVTDHGSCVRAC GADSYEMEEDGVRKC KKCEGPCRKVCNGIGI GEFKD EGFRS EGFR:SEPT1LPQPPICTIDVYMIMV 1QLQDKFEHL (A02.01, GBM, Glioma, EPT14 4KCWMIDADSRPKFRE B08.01) Head and Neck LIIEFSKMARDPQRYLQLQDKFEHL (A02.01, Cancer VIQ::LQDKFEHLKMI B08.01) QQEEIRKLEEEKKQLQLQDKFEHLK (A03.01) EGEIIDFYKMKAASE YLVIQLQDKF (A02.01, alqtqlstdA24.02) EML4:A EML4:ALK SWENSDDSRNKLSKIP QVYRRKHQEL (B08.01) NSCLC LKSTPKLIPKVTKTADKH STREKNSQV (B08.01) KDVIINQAKMSTREK VYRRKHQEL (A24.02,NSQ:V:YRRKHQELQ B08.01) AMQMELQSPEYKLS KLRTSTIMTDYNPNY CFAGKTSSISDLFGFR3: FGFR3:TACC EGHRMDKPANCTHDL VLTVTSTDV (A02.01) Bladder Cancer,TACC3 3 YMIMRECWHAAPSQR VLTVTSTDVK (A03.01) LUSC PTFKQLVEDLDRVLTVTSTD::VKATQEENR ELRSRCEELHGKNLE LGKIMDRFEEVVYQ AMEEVQKQKELS NAB:STNAB:STAT6 RDNTLLLRRVELFSLS IMSLWGLVS (A02.01) Solitary fibrous AT6 “”RQVARESTYLSSLKGS IMSLWGLVSK (A03.01) tumors RLHPEELGGPPLKKLKKLKQEATSK (A03.01) QE::ATSKSQI MSLVV QIMSLWGLV (A02.01) GLVSKMPPEKVQRLYSQIMSLWGL (A02.01, VDFPQHLRHLLGDW A24.02, B08.01) LESQPWEFLVGSDAFSQ1MSLWGLV (A02.01) CC TSKSQIMSL (B08.01) NDRG1: NDRG1:ERGMSREMQDVDLAEVKP LLQEFDVQEA (A02.01) Prostate Cancer ERG LVEKGETITGLLQEFDLQEFDVQEAL (A02.01) VQ::EALSVVSEDQSL FECAYGTPHLAKTE MTASSSSDYGQTSKMSPRVPQQDW PMLRA PMLRARA VLDMHGFLRQALCRL Acute RA (exon3:RQEEPQSLQAAVRTDG promyelocytic exon3) FDEFKVRLQDLSSCIT leukemiaQGK:A:IETQSSSSEE IVPSPPSPPPLPRIYK PCFVCQDKSSGYHYG VSACEGCKG PMLRAPMLRARA RSSPEQPRPSTSKAVSP Acute RA (exon6: PHLDGPPSPRSPVIGSEpromyelocytic exon3) VFLPNSNHVASGAGEA: leukemia A:IETQSSSSEEIVPSPPSPPPLPRIYKPCFV CQDKSSGYHYGVSA CEGCKG RUNX1 RUNX1(ex5)- VARFNDLRFVGRSGRGPREPRNRT (B07.02) AML RUNX1T1 GKSFTLTITVFTNPPQ RNRTEKHSTM (B08.01)(ex2) VATYHRAIKITVDGPR EPR:N:RTEKHSTMPD SPVDVKTQSRLTPPT MPPPPTTQCAPRTSSFTPTTLTNGT TMPRS TMPRSS2: MALNS::EALSVVSED ALNSEALSV (A02.01)Prostate Cancer S2ERG ERG QSLFECAYGTPHLAKT ALNSEALSVV (A02.01)EMTASSSSDYGQTSK MALNSEALSV (A02.01, MSPRVPQQDW B08.01) ¹ Underlined AAsrepresent non-native AAs ² Bolded AAs represent native AAs of the aminoacid sequence encoded by the second of the two fused genes ³ Bolded andunderlined AAs represent non-native AAs of the amino acid sequenceencoded by the second of the two fused genes due to a frameshift.

TABLE 2 Exemplary Protein Mutaation Sequence Peptides (HLA allele GeneChange Context example(s)) Exemplary Diseases Table 2A POINT MUTATIONS¹AKT1 E17K MSDVAIVKEGWLHKR KYIKTWRPRY (A24.02) BRCA, CESC, HNSC,GKYIKTWRPRYFLLK WLHKRGKYI (A02.01, B07.02, LUSC, PRAD, SKCM,NDGTFIGYKERPQDV B08.01) THCA DQREAPLNNFSVAQC WLHKRGKYIK (A03.01) QLMKTERANAPC1 T537A TMLVLEGSGNLVLYT APKPLSKLL (B07.02) GBM, LUSC, PAAD,GVVRVGKVFIPGLPAP GVSAPKPLSK (A03.01) PRAD, SKCM SLTMSNTMPRPSTPLDVSAPKPLSK (A03.01) GVSAPKPLSKLLGSLD EVVLLSPVPELRDSSK LHDSLYNEDCTFQQLGTYIHSI FGFR3 S249C HRIGGIKLRHQQWSL CPHRPILQA (B07.02) BLCA, HNSC, KIRP,VMESVVPSDRGNYTC LUSC VVENKFGSIRQTYTLD VLERCPHRPILQAGLP ANQTAVLGSDVEFHCKVYSDAQPHIQWLKH VEVNGSKVG FRG1B I10T MREPIYMHSTMVFLPKLSDSRTAL (A02.01, B07.02, KIRP, PRAD, SKCM WELHTKKGPSPPEQF B08.01)MAVKLSDSRTALKSG KLSDSRTALK (A03.01) YGKYLGINSDELVGHLSDSRTALK (A01.01, A03.01) SDAIGPREQWEPVFQ RTALKSGYGK (A03.01)NGKMALLASNSCFIR TALKSGYGK (A03.01) FRG1B L52S AVKLSDSRIALKSGYGALSASNSCF (A02.01, A24.02, GBM, KIRP, PRAD, KYLGINSDELVGHSD B07.02) SKCMAIGPREQWEPVFQNG ALSASNSCFI (A02.01) KMALSASNSCFIRCNE FQNGKMALSA (A02.01,AGDIEAKSKTAGEEE B08.01) MIKIRSCAEKETKKKD DIPEEDKG HER2 L755SAMPNQAQMRILKETE KVSRENTSPK (A03.01) BRCA (Resistance) LRKVKVLGSGAFGTVYKGIWIPDGENVKIPV AIKVSRENTSPKANKE ILDEAYVMAGVGSPY VSRLLGICLTSTVQLVTQLMPYGC IDH1 R132G RVEEFKLKQMWKSPN KPIIIGGHAY (B07.02) BLCA, BRCA, CRC,GTIRNILGGTVFREAII GBM, HNSC, LUAD, CKNIPRLVSGWVKPIII PAAD, PRAD, UCECGGHAYGDQYRATDF VVPGPGKVEITYTPSD GTQKVTYLVHNFEEG GGVAMGM KRAS G12CMTEYKLVVVGACGV KLVVVGACGV (A02.01) BRCA, CESC, CRC, GKSALTIQLIQNHFVDLVVVGACGV (A02.01) HNSC, LUAD, PAAD, EYDPTIEDSYRKQVVI VVGACGVGK (A03.01,UCEC DGETCLLDILDTAGQE A11.01) VVVGACGVGK (A03.01) KRAS Gl2DMTEYKLVVVGADGV VVGADGVGK (A11.01) BLCA, BRCA, CESC, GKSALTIQLIQNHFVDVVVGADGVGK (A11.01) CRC, GBM, HNSC, EYDPTIEDSYRKQVVI KLVVVGADGV (A02.01)KIRP, LIHC, LUAD, DGETCLLDILDTAGQE LVVVGADGV (A02.01) PAAD, SKCM, UCECKRAS G12V MTEYKLVVVGAVGV KLVVVGAVGV (A02.01) BRCA, CESC, CRC,GKSALTIQLIQNHFVD LVVVGAVGV (A02.01) LUAD, PAAD, THCA, EYDPTIEDSYRKQVVIVVGAVGVGK (A03.01, UCEC DGETCLLDILDTAGQE A11.01) VVVGAVGVGK (A03.01,A11.01) KRAS Q61H AGGVGKSALTIQLIQN ILDTAGHEEY (A01.01) CRC, LUSC, PAAD,HFVDEYDPTIEDSYRK SKCM, UCEC QVVIDGETCLLDILDT AGHEEYSAMRDQYMRTGEGFLCVFAINNTK SFEDIHHYREQIKRVK DSEDVPM SF3B1 K700E AVCKSKKSWQARHTGLVDEQQEV (A02.01) AML associated with GIKIVQQIAILMGCAIL MDS; ChronicPHLRSLVEIIEHGLVD lymphocytic leukemia- EQQEVRTISALAIAALsmall lymphocytic AEAATPYGIESFDSVL lymphoma; KPLWKGIRQHRGKGLMyelodysplastic AAFLKAI syndrome; AML; Luminal NS carcinoma of breast;Chronic myeloid leukemia; Ductal carcinoma of pancreas;Chronic myelomonocytic leukemia; Chronic lymphocytic leukemia-small lymphocytic lymphoma; Myelofibrosis; Myelodysplasticsyndrome; PRAD; Essential thrombocythaemia; Medullomyoblastoma SPOPF133L YLSLYLLLVSCPKSEV FVQGKDWGL (A02.01 PRAD RAKFKFSILNAKGEET B08.01)KAMESQRAYRFVQG KDWGLKKFIRRDFLL DEANGLLPDDKLTLF CEVSVVQDSVNISGQNTMNMVKVPE SPOP F133V YLSLYLLLVSCPKSEV FVQGKDWGV (A02.01) PRADRAKFKFSILNAKGEET KAMESQRAYRFVQG KDWGVKKFIRRDFLL DEANGLLPDDKLTLFCEVSVVQDSVNISGQ NTMNMVKVPE TP53 G245S IRVEGNLRVEYLDDRCMGSMNRRPI (A02.01, BLCA, BRCA, CRC, NTFRHSVVVPYEPPEV B08.01)GBM, HNSC, LUSC, GSDCTTIHYNYMCNS GSMNRRPIL (B08.01) PAAD, PRADSCMGSMNRRPILTIITL MGSMNRRPI (B08.01) EDSSGNLLGRNSFEVRMGSMNRRPIL (B08.01) VCACPGRDRRTEEEN SMNRRPILTI (A02.01, A24.02, LRKKGEPB08.01) TP53 R248Q EGNLRVEYLDDRNTF CMGGMNQRPI (A02.01, BLCA, BRCA, CRC,RHSVVVPYEPPEVGS B08.01) GBM, HNSC, KIRC, DCTTIHYNYMCNSSCGMNQRPILTI (A02.01, B08.01) LIHC, LUSC, PAAD, MGGMNQRPILTIITLENQRPILTII (A02.01, B08.01) PRAD, UCEC DSSGNLLGRNSFEVR VCACPGRDRRTEEENLRKKGEPHHE TP53 R248W EGNLRVEYLDDRNTF CMGGMNWRPI (A02.01,BLCA, BRCA, CRC, RHSVVVPYEPPEVGS A24.02, B08.01) GBM, HNSC, LIHC,DCTTIHYNYMCNSSC GMNWRPILTI (A02.01, LUSC, PAAD, SKCM, MGGMNWRPILTIITLEB08.01) UCEC DSSGNLLGRNSFEVR MNWRPILTI (A02.01, A24.02, VCACPGRDRRTEEENB08.01) LRKKGEPHHE MNWRPILTII (A02.01 A24.02) TP53 R273C PEVGSDCTTIHYNYMNSFEVCVCA (A02.01) BLCA, BRCA, CRC, CNSSCMGGMNRRPIL GBM, HNSC, LUSC,TIITLEDSSGNLLGRNS PAAD, UCEC FEVCVCACPGRDRRT EEENLRKKGEPHHELPPGSTKRALPNNTSSSP QPKKKPL TP53 R273H PEVGSDCTTIHYNYM BRCA, CRC, GBM,HNSC, LIHC, LUSC, CNSSCMGGMNRRPIL NSFEVHVCA (A02.01) TIITLEDSSGNLLGRNSPAAD, UCEC FEVHVCACPGRDRRT EEENLRKKGEPHHELP PGSTKRALPNNTSSSP QPKKKPLTP53 Y220C TEVVRRCPHHERCSD VVPCEPPEV (A02.01) BLCA, BRCA, GBM,SDGLAPPQHLIRVEGN VVVPCEPPEV  HNSC, LIHC, LUAD, LRVEYLDDRNTFRHS (A02.01)LUSC, PAAD, SKCM, VVVPCEPPEVGSDCTT UCEC IHYNYMCNSSCMGG MNRRPILTIITLEDSSGNLLGRNSF Table 2B MSI-ASSOCIATED FRAMESHIFTS¹ MSH6 F1088fs; +1YNFDKNYKDWQSAV ILLPEDTPPL (A02.01) MSI+ CRC, MSI+ ECIAVLDVLLCLANYSLLPEDTPPL (A02.01) Uterine/Endometrium RGGDGPMCRPVILLPECancer, MSI+ Stomach DTPPLLRA Cancer, Lynch syndromeTable 2C FRAMESHIFT¹ APC F1354fs AKFQQCHSTLEPNPA APFRVNHAV (B07.02)CRC, LUAD, UCEC, DCRVLVYLQNQPGTK CLADVLLSV (A02.01) STADLLNFLQERNLPPKVVL FLQERNLPPK (A03.01) RHPKVHLNTMFRRPH HLIVLRVVRL (A02.01,SCLADVLLSVHLIVLR B08.01) VVRLPAPFRVNHAVE HPKVHLNTM (B07.02, W* B08.01)HPKVHLNTMF (B07.02, B08.01) KVHLNTMFR (A03.01) KVHLNTMFRR (A03.01)LPAPFRVNHA (B07.02) MFRRPHSCL (B07.02, B08.01) MFRRPHSCLA (B08.01)NTMFRRPHSC (B08.01) RPHSCLADV (B07.02) RPHSCLADVL (B07.02)RVVRLPAPFR (A03.01) SVHLIVLRV (A02.01) TMFRRPHSC (B08.01)TMFRRPHSCL (A02.01, B08.01) VLLSVHLIV (A02.01) VLLSVHLIVL (A02.01)VLRVVRLPA (B08.01) VVRLPAPFR (A03.01) ARID1A Y1324fs ALGPHSRISCLPTQTRAMPILPLPQL (A02.01) STAD, UCEC, BLCA, GCILLAATPRSSSSSSSAPLLAAPSPA (B07.02) BRCA, LUSC, CESC, NDMIPMAISSPPKAPLAPRTNFHSS (B07.02) KIRC, UCS LAAPSPASRLQCINSN APRTNFHSSL (B07.02,SRITSGQWMAHMALL B08.01) PSGTKGRCTACHTAL CPQPSPSLPA (B07.02)GRGSLSSSSCPQPSPSL GQWMAHMAL (A02.01) PASNKLPSLPLSKMYTGQWMAHMALL (A02.01) TSMAMPILPLPQLLLS HMALLPSGTK (A03.01) ADQQAAPRTNFHSSLHTALGRGSL (B07.02) AETVSLHPLAPMPSKT IPMAISSPP (B07.02) CHHK*IPMAISSPPK (B07.02) KLPSLPLSK (A03.01) KLPSLPLSKM (A02.01)KMYTTSMAM (A02.01, A03.01) LLAAPSPASR (A03.01) LLLSADQQAA (A02.01)LLSADQQAA (A02.01) LPASNKLPS (B07.02) LPASNKLPSL (B07.02, B08.01)LPLPQLLLSA (B07.02) LPSLPLSKM (B07.02) LSKMYTTSM (B08.01)MALLPSGTK (A03.01) MPILPLPQL (B07.02) MPILPLPQLL (B07.02)MYTTSMAMPI (A24.02) PMAISSPPK (A03.01) QWMAHMALL (A24.02)SKMYTTSMAM (B07.02) SMAMPILPL (A02.01, B07.02, B08.01)SNKLPSLPL (B08.01) SPASRLQCI (B07.02, B08.01) SPPKAPLLAA (B07.02)SPSLPASNKL (B07.02) YTTSMAMPI (A02.01) YTTSMAMPIL (A02.01) ARID1AG1848fs RSYRRMIHLWWTAQI CLPGLTHPA (A02.01) STAD, UCEC, BLCA,SLGVCRSLTVACCTG GLTHPAHQPL (A02.01) BRCA, LUSC, CESC, GLVGGTPLSISRPTSRHPAHQPLGSM (B07.02) KIRC, UCS ARQSCCLPGLTHPAH LTHPAHQPL (B07.02) QPLGSM*RPTSRARQSC (B07.02) RQSCCLPGL (A02.01) TSRARQSCCL (B08.01) β2M L13fsQHSGRDVSLRGLSCA ELLCVWVSSI (A02.01) CRC, STAD, SKCM, RATLSFWPGGYPAYSEWKVKFPEL (B08.01) HNSC KDSGLLTSSSREWKV KFPELLCVW (A24.02)KFPELLCVWVSSIRH* LLCVWVSSI (A02.01) LLTSSSREWK (A03.01)LTSSSREWK (A03.01) YPAYSKDSGL (B07.02) GATA3 L328fs AQAKAVCSQESRDVLCLQCLWALL (A02.01) Breast Cancer N334fs CELSDFIHNHTLEEECCQWGPCLQCL (A02.01) QWGPCLQCLWALLQ QWGPCLQCL (A24.02) ASQY*QWGPCLQCLW (A24.02) GATA3 H400fs PGRPLQTHVLPEPHLA AIQPVLWTT (A02.01)Breast Cancer S408fs LQPLQPHADHAHADA ALQPLQPHA (A02.01) S408fsPAIQPVLWTTPPLQHG DLHFCRSSIM (B08.01) S430fs HRHGLEPCSMLTGPPEPHLALQPL (B07.02, B08.01) H434fs ARVPAVPFDLHFCRSS ESKIMFATL (B08.01)H435fs IMKPKRDGYMFLKAE FATLQRSSL (B07.02, B08.01) SKIMFATLQRSSLWCLFLKAESKIM (B08.01) CSNH* FLKAESKIMF (B08.01) GPPARVPAV (B07.02)IMKPKRDGYM (B08.01) KIMFATLQR (A03.01) KPKRDGYMF (B07.02)KPKRDGYMFL (B07.02) LHFCRSSIM (B08.01) LQHGHRHGL (B08.01)MFATLQRSSL (B07.02, B08.01) MFLKAESKI (A24.02) MLTGPPARV (A02.01)QPVLWTTPPL (B07.02) SMLTGPPARV (A02.01) TLQRSSLWCL (A02.01)VLPEPHLAL (A02.01) VPAVPFDLHF (B07.02) YMFLKAESK (A03.01)YMFLKAESKI (A02.01, A03.01, A24.02, B08.01) MLL2 P647fs TRRCHCCPHLRSHPCPAPGPRGRTC (B07.02) STAD, BLCA, CRC, L656fs HHLRNHPRPHHLRHHCLRSHTCPPR (A03.01) HNSC, BRCA ACHHHLRNCPHPHFL CLWCHACLHR (A03.01)RHCTCPGRWRNRPSL CPHLGSHPC (B07.02) RRLRSLLCLPHLNHHL CPLGLKSPL (B07.02)FLHWRSRPCLHRKSH CPRSCRCPH (B07.02) PHLLHLRRLYPHHLK CPRSCRCPHL (B07.02,HRPCPHHLKNLLCPR B08.01) HLRNCPLPRHLKHLA CSLPLGNHPY (A01.01)CLHHLRSHPCPLHLKS GLRNRICPL (A02.01, B07.02, HPCLHHRRHLVCSHH B08.01)LKSLLCPLHLRSLPFP GLRSHTYLR (A03.01) HHLRHHACPHHLRTR GLRSHTYLRR (A03.01)LCPHHLKNHLCPPHL GPRGRTCHPG (B07.02) RYRAYPPCLWCHACL HLGSHPCRL (B08.01)HRLRNLPCPHRLRSLP HLRLHASPH (A03.01) RPLHLRLHASPHHLRTHLRSCPCSL (B07.02, B08.01) PPHPHHLRTHLLPHHR HLRTHLLPH (A03.01)RTRSCPCRWRSHPCC HLRTHLLPHH (A03.01) HYLRSRNSAPGPRGR HLRYRAYPP (B08.01)TCHPGLRSRTCPPGLR HLRYRAYPPC (B08.01) SHTYLRRLRSHTCPPS HPHHLRTHL (B07.02)LRSHAYALCLRSHTCP HPHHLRTHLL (B07.02, PRLRDHICPLSLRNCT B08.01)CPPRLRSRTCLLCLRS HTYLRRLRSH (A03.01) HACPPNLRNHTCPPSLLPCPHRLRSL (B07.02, B08.01) RSHACPPGLRNRICPL LPHHRRTRSC (B07.02,SLRSHPCPLGLKSPLR B08.01) SQANALHLRSCPCSLP LPLGNHPYL (B07.02)LGNHPYLPCLESQPCL LPRPLHLRL (B07.02, B08.01) SLGNHLCPLCPRSCRCNLRNHTCPP (B08.01) PHLGSHPCRLS* PPRLRSRTCL (B07.02, B08.01)RLHASPHHL (A02.01) RLHASPHHLR (A03.01) RLRDHICPL (A02.01, B07.02,B08.01) RLRNLPCPH (A03.01) RLRNLPCPHR (A03.01) RLRSHTCPP (B08.01)RLRSLPRPL (B07.02, B08.01) RLRSLPRPLH (A03.01)RLRSRTCLL (B07.02, B08.01) RNRICPLSL (B07.02, B08.01) RPLHLRLHA (B07.02)RPLHLRLHAS (B07.02) RSHACPPGLR (A03.01) RSHACPPNLR (A03.01)RSHAYALCLR (A03.01) RSHPCCHYLR (A03.01) RSHPCPLGLK (A03.01)RSHTCPPSLR (A03.01) RSLPRPLHLR (A03.01) RSRTCLLCL (B07.02)RSRTCLLCLR (A03.01) RSRTCPPGL (B07.02) RSRTCPPGLR (A03.01)RTHLLPHHRR (A03.01) RTRSCPCRWR (A03.01) RYRAYPPCL (A24.02) MLL2 P2354fsGPRSHPLPRLWHLLL ALAPTLTHM (A02.01) STAD, BLCA, CRC, QVTQTSFALAPTLTHALAPTLTHML (A02.01) HNSC, BRCA MLSPH* LLQVTQTSFA (A02.01)LQVTQTSFAL (A02.01) RLWHLLLQV (A02.01) RLWHLLLQVT (A02.01) RNF43 G659fsPLGLVPWTRWCPQGK CTQLARFFPI (A24.02) STAD PRFPAMSTTTATGTTTFFPITPPVW (A24.02) TKSGSSGMAGSLAQK FPITPPVWHI (B07.02) PESPSPGLLFLGHSPSQGPRMQLCTQL (B07.02, SHLLLISKSPDPTQQPL B08.01) RGGSLTHSAPGPSLSQITPPVWHIL (A24.02) PLAQLTPPASAPVPAV LALGPRMQL (B07.02) CSTCKNPASLPDTHRGMQLCTQLARF (A24.02) KGGGVPPSPPLALGPR RFFPITPPV (A02.01, A24.02)MQLCTQLARFFPITPP RFFPITPPVW (A24.02) VWHILGPQRHTP* RMQLCTQLA (A02.01)RMQLCTQLAR (A03.01) SPPLALGPRM (B07.02) TQLARFFPI (A02.01, A24.02,B08.01) SMAP1 E169fs KYEKKKYYDKNAIAI KSRQNHLQL (B07.02) MSI+ CRC, MSI+TNISSSDAPLQPLVSSP ALKKLRSPL (B08.01, B07.02) Uterine/EndometriumSLQAAVDKNKLEKEK HLQLKSCRRK (A03.01) Cancer, MSI+ Stomach EKKRKRKREKRSQKSKISNWSLKK (A03.01, A11.01) Cancer RQNHLQLKSCRRKISN KISNWSLKKV (A03.01)WSLKKVPALKKLRSP KLRSPLWIF (A24.02) LWIF KSRQNHLQLK (A03.01)NWSLKKVPAL (B08.01) SLKKVPALK (A03.01, A11.01) SLKKVPALKK (A03.01)SQKSRQNHL (B08.01) WSLKKVPAL (B08.01) WSLKKVPALK (A03.01) TP53 P58fsCCPRTILNNGSLKTQV KLPECQRLL (A02.01) BRCA, CRC, LUAD, P72fsQMKLPECQRLLPPWP KPTRAATVSV (B07.02) PRAD, HNSC, LUSC, G108fsLHQQLLHRRPLHQPPP LPPWPLHQQL (B07.02) PAAD, STAD, BLCA, R110fsGPCHLLSLPRKPTRAA LPRKPTRAA (B07.02, B08.01) OV, LIHC, SKCM,TVSVWASCILGQPSL* LPRKPTRAAT (B07.02) UCEC, LAML, UCS, QQLLHRRPL (B08.01)KICH, GBM, ACC RLLPPWPLH (A03.01) TP53 P152fs LARTPLPSTRCFANWPAPASAPWPST (B07.02) BRCA, CRC, LUAD, RPALCSCGLIPHPRPA APWPSTSSH (B07.02)PRAD, HNSC, LUSC, PASAPWPSTSSHST* RPAPASAPW (B07.02) PAAD, STAD, BLCA,WPSTSSHST (B07.02) OV, LIHC, SKCM, UCEC, LAML, UCS, KICH, GBM, ACC UBR5K2120fs SQGLYSSSASSGKCL RVQNQGHLL (B07.02) MEVTVDRNCLEVLPTKMSYAANLKNVMNM QNRQKKKGKNSPCCQ KKLRVQNQGHLLMIL LHN* VHL L116fsTRASPPRSSSAIAVRA FLPISHCQCI (A02.01) KIRC, KIRP G123fs SCCPYGSTSTASRSPTFWLTKLNYL (A24.02, B08.01) ORCRLARAAASTATE HLSMLTDSL (A02.01)VTFGSSEMQGHTMGF HTMGFWLTK (A03.01) WLTKLNYLCHLSMLT HTMGFWLTKL (A02.01)DSLFLPISHCQCIL* KLNYLCHLSM (A02.01) LPISHCQCI (B07.02, B08.01)LPISHCQCIL (B07.02, B08.01) LTDSLFLPI (A01.01, A02.01)LTKLNYLCHL (B08.01) MLTDSLFLPI (A01.01, A02.01, B08.01)MQGHTMGFWL (A02.01) NYLCHLSML (A24.02) SMLTDSLFL (A02.01)TMGFWLTKL (A02.01) YLCHLSMLT (A02.01) TABLE 2D INSERT¹ HER2 G776insYVMALGSGAFGTVYKGIWIP ILDEAYVMAY (A01.01) Lung Cancer DGENVKIPVAIKVLREVMAYVMAGV (A02.01) NTSPKANKEILDEAYV YVMAYVMAG (A02.01, MAYVMAGVGSPYVSB07.02, B08.01) RLLGICLTSTVQLVTQ YVMAYVMAGV (A02.01, LMPYGCLLDHVRENRB07.02, B08.01) GRLGSQDLLNW ¹Underlined AAs represent non-native AAs²Bolded AAs represent native AAs of the amino acid sequence encoded bythe second of the two fused genes ³Bolded and underlined AAs representnon-native AAs of the amino acid sequence encoded by the second of thetwo fused genes due to a frameshift.

In the Tables above, for one or more of the exemplary fusions, asequence that comes before the first “:” belongs to an exon sequence ofa polypeptide encoded by a first gene, a sequence that comes after thesecond “:” belongs to an exon sequence of a polypeptide encoded by asecond gene, and an amino acid that appears between “:” symbols isencoded by a codon that is split between the exon sequence of apolypeptide encoded by a first gene and the exon sequence of apolypeptide encoded by a second gene.

However, in some embodiments, for example, NAB:STAT6, the NAB exon islinked to the 5′ UTR of STAT6 and the first amino acid that appearsafter the junction is the normal start codon of STAT6 (there is no framepresent at this site (as it is not normally translated).

AR-V7 in the tables above can also be considered, in some embodiments, asplice variant of the AR gene that encodes a protein that lacks theligand binding domain found in full length AR.

In some embodiments, sequencing methods are used to identify tumorspecific mutations. Any suitable sequencing method can be used accordingto the present disclosure, for example, Next Generation Sequencing (NGS)technologies. Third Generation Sequencing methods might substitute forthe NGS technology in the future to speed up the sequencing step of themethod. For clarification purposes: the terms “Next GenerationSequencing” or “NGS” in the context of the present disclosure mean allnovel high throughput sequencing technologies which, in contrast to the“conventional” sequencing methodology known as Sanger chemistry, readnucleic acid templates randomly in parallel along the entire genome bybreaking the entire genome into small pieces. Such NGS technologies(also known as massively parallel sequencing technologies) are able todeliver nucleic acid sequence information of a whole genome, exome,transcriptome (all transcribed sequences of a genome) or methylome (allmethylated sequences of a genome) in very short time periods, e.g.within 1-2 weeks, for example, within 1-7 days or within less than 24hours and allow, in principle, single cell sequencing approaches.Multiple NGS platforms which are commercially available or which arementioned in the literature can be used in the context of the presentdisclosure e.g. those described in detail in WO 2012/159643.

In certain embodiments, the peptide described herein can comprise, butis not limited to, about 5, about 6, about 7, about 8, about 9, about10, about 11, about 12, about 13, about 14, about 15, about 16, about17, about 18, about 19, about 20, about 21, about 22, about 23, about24, about 25, about 26, about 27, about 28, about 29, about 30, about31, about 32, about 33, about 34, about 35, about 36, about 37, about38, about 39, about 40, about 41, about 42, about 43, about 44, about45, about 46, about 47, about 48, about 49, about 50, about 60, about70, about 80, about 90, about 100, about 110, about 120, about 150,about 200, about 300, about 350, about 400, about 450, about 500, about600, about 700, about 800, about 900, about 1,000, about 1,500, about2,000, about 2,500, about 3,000, about 4,000, about 5,000, about 7,500,about 10,000 amino acids or greater amino acid residues, and any rangederivable therein. In specific embodiments, a neoantigenic peptidemolecule is equal to or less than 100 amino acids.

In some embodiments, the peptides can be from about 8 and about 50 aminoacid residues in length, or from about 8 and about 30, from about 8 andabout 20, from about 8 and about 18, from about 8 and about 15, or fromabout 8 and about 12 amino acid residues in length. In some embodiments,the peptides can be from about 8 and about 500 amino acid residues inlength, or from about 8 and about 450, from about 8 and about 400, fromabout 8 and about 350, from about 8 and about 300, from about 8 andabout 250, from about 8 and about 200, from about 8 and about 150, fromabout 8 and about 100, from about 8 and about 50, or from about 8 andabout 30 amino acid residues in length.

In some embodiments, the peptides can be at least 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, or more amino acid residues in length. In some embodiments, thepeptides can be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90,100, 150, 200, 250, 300, 350, 400, 450, 500, or more amino acid residuesin length. In some embodiments, the peptides can be at most 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, or less amino acid residues in length. In someembodiments, the peptides can be at most 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55,60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or lessamino acid residues in length.

In some embodiments, the peptides has a total length of at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, at least 22, at least 23, at least 24, at least25, at least 26, at least 27, at least 28, at least 29, at least 30, atleast 40, at least 50, at least 60, at least 70, at least 80, at least90, at least 100, at least 150, at least 200, at least 250, at least300, at least 350, at least 400, at least 450, or at least 500 aminoacids.

In some embodiments, the peptides has a total length of at most 8, atmost 9, at most 10, at most 11, at most 12, at most 13, at most 14, atmost 15, at most 16, at most 17, at most 18, at most 19, at most 20, atmost 21, at most 22, at most 23, at most 24, at most 25, at most 26, atmost 27, at most 28, at most 29, at most 30, at most 40, at most 50, atmost 60, at most 70, at most 80, at most 90, at most 100, at most 150,at most 200, at most 250, at most 300, at most 350, at most 400, at most450, or at most 500 amino acids.

A longer peptide can be designed in several ways. In some embodiments,when HLA-binding peptides are predicted or known, a longer peptidecomprises (1) individual binding peptides with extensions of 2-5 aminoacids toward the N- and C-terminus of each corresponding gene product;or (2) a concatenation of some or all of the binding peptides withextended sequences for each. In other embodiments, when sequencingreveals a long (>10 residues) neoepitope sequence present in the tumor(e.g., due to a frameshift, read-through or intron inclusion that leadsto a novel peptide sequence), a longer peptide could consist of theentire stretch of novel tumor-specific amino acids as either a singlelonger peptide or several overlapping longer peptides. In someembodiments, use of a longer peptide is presumed to allow for endogenousprocessing by patient cells and can lead to more effective antigenpresentation and induction of T cell responses. In some embodiments, twoor more peptides can be used, where the peptides overlap and are tiledover the long neoantigenic peptide.

In some embodiments, the peptides can have a pI value of from about 0.5to about 12, from about 2 to about 10, or from about 4 to about 8. Insome embodiments, the peptides can have a pI value of at least 4.5, 5,5.5, 6, 6.5, 7, 7.5, or more. In some embodiments, the peptides can havea pI value of at most 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or less.

In some embodiments, the peptide described herein can be in solution,lyophilized, or can be in crystal form. In some embodiments, the peptidedescribed herein can be prepared synthetically, by recombinant DNAtechnology or chemical synthesis, or can be isolated from naturalsources such as native tumors or pathogenic organisms. Neoepitopes canbe synthesized individually or joined directly or indirectly in thepeptide. Although the peptide described herein can be substantially freeof other naturally occurring host cell proteins and fragments thereof,in some embodiments, the peptide can be synthetically conjugated to bejoined to native fragments or particles.

In some embodiments, the peptide described herein can be prepared in awide variety of ways. In some embodiments, the peptides can besynthesized in solution or on a solid support according to conventionaltechniques. Various automatic synthesizers are commercially availableand can be used according to known protocols. See, for example, Stewart& Young, Solid Phase Peptide Synthesis, 2d. Ed., Pierce Chemical Co.,1984. Further, individual peptides can be joined using chemical ligationto produce larger peptides that are still within the bounds of thepresent disclosure.

Alternatively, recombinant DNA technology can be employed wherein anucleotide sequence which encodes the peptide inserted into anexpression vector, transformed or transfected into an appropriate hostcell and cultivated under conditions suitable for expression. Theseprocedures are generally known in the art, as described generally inSambrook et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (1989). Thus, recombinantpeptides, which comprise one or more neoantigenic peptides describedherein, can be used to present the appropriate T cell epitope.

In some embodiments, the peptide is encoded by a gene with a pointmutation resulting in an amino acid substitution of the native peptide.In some embodiments, the peptide is encoded by a gene with a pointmutation resulting in frame shift mutation. A frameshift occurs when amutation disrupts the normal phase of a gene's codon periodicity (alsoknown as “reading frame”), resulting in the translation of a non-nativeprotein sequence. It is possible for different mutations in a gene toachieve the same altered reading frame. In some embodiments, the peptideis encoded by a gene with a mutation resulting in fusion polypeptide,in-frame deletion, insertion, expression of endogenous retroviralpolypeptides, and tumor-specific overexpression of polypeptides. In someembodiments, the peptide is encoded by a fusion of a first gene with asecond gene. In some embodiments, the peptide is encoded by an in-framefusion of a first gene with a second gene. In some embodiments, thepeptide is encoded by a fusion of a first gene with an exon of a splicevariant of the first gene. In some embodiments, the peptide is encodedby a fusion of a first gene with a cryptic exon of the first gene. Insome embodiments, the peptide is encoded by a fusion of a first genewith a second gene, wherein the peptide comprises an amino acid sequenceencoded by an out of frame sequence resulting from the fusion.

In some aspects, the present disclosure provides a compositioncomprising at least two or more than two peptides. In some embodiments,the composition described herein contains at least two distinctpeptides. In some embodiments, the composition described herein containsa first peptide comprising a first neoepitope and a second peptidecomprising a second neoepitope. In some embodiments, the first andsecond peptides are derived from the same protein. The at least twodistinct peptides may vary by length, amino acid sequence or both. Thepeptides can be derived from any protein known to or have been found tocontain a tumor specific mutation. In some embodiments, the compositiondescribed herein comprises a first peptide comprising a first neoepitopeof a protein and a second peptide comprising a second neoepitope of thesame protein, wherein the first peptide is different from the secondpeptide, and wherein the first neoepitope comprises a mutation and thesecond neoepitope comprises the same mutation. In some embodiments, thecomposition described herein comprises a first peptide comprising afirst neoepitope of a first region of a protein and a second peptidecomprising a second neoepitope of a second region of the same protein,wherein the first region comprises at least one amino acid of the secondregion, wherein the first peptide is different from the second peptideand wherein the first neoepitope comprises a first mutation and thesecond neoepitope comprises a second mutation. In some embodiments, thefirst mutation and the second mutation are the same. In someembodiments, the mutation is selected from the group consisting of apoint mutation, a splice-site mutation, a frameshift mutation, aread-through mutation, a gene fusion mutation and any combinationthereof.

In some embodiments, the peptide can be derived from a protein with asubstitution mutation, e.g., the KRAS G12C, G12D, G12V, Q61H or Q61Lmutation, or the NRAS Q61K or Q61R mutation. The substitution may bepositioned anywhere along the length of the peptide. For example, it canbe located in the N terminal third of the peptide, the central third ofthe peptide or the C terminal third of the peptide. In anotherembodiment, the substituted residue is located 2-5 residues away fromthe N terminal end or 2-5 residues away from the C terminal end. Thepeptides can similarly derived from tumor specific insertion mutationswhere the peptide comprises one or more, or all of the insertedresidues.

In some embodiments, the first peptide comprises at least one anadditional mutation. In some embodiments, one or more of the at leastone additional mutation is not a mutation in the first neoepitope. Insome embodiments, one or more of the at least one additional mutation isa mutation in the first neoepitope. In some embodiments, the secondpeptide comprises at least one additional mutation. In some embodiments,one or more of the at least one additional mutation is not a mutation inthe second neoepitope. In some embodiments, one or more of the at leastone additional mutation is a mutation in the second neoepitope.

In some aspects, the present disclosure provides a compositioncomprising a single polypeptide comprises the first peptide and thesecond peptide, or a single polynucleotide encodes the first peptide andthe second peptide. In some embodiments, the composition provided hereincomprises one or more additional peptides, wherein the one or moreadditional peptides comprise a third neoepitope. In some embodiments,the first peptide and the second peptide are encoded by a sequencetranscribed from the same transcription start site. In some embodiments,the first peptide is encoded by a sequence transcribed from a firsttranscription start site and the second peptide is encoded by a sequencetranscribed from a second transcription start site. In some embodiments,wherein the polypeptide has a length of at least 26; 27; 28; 29; 30; 40;50; 60; 70; 80; 90; 100; 150; 200; 250; 300; 350; 400; 450; 500; 600;700; 800; 900; 1,000; 1,500; 2,000; 2,500; 3,000; 4,000; 5,000; 7,500;or 10,000 amino acids. In some embodiments, the polypeptide comprises afirst sequence with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74% 75% 76%, 77% 78% 79%, 80%, 81%, 82%,83% 84%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity to a corresponding wild-type sequence; anda second sequence with at least 60%, 61%, 62%, 63%, 64%, 65%, 66% 67%,68% 69%, 70%, 71%, 72%, 73% 74% 75%, 76% 77% 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to a corresponding wild-typesequence. In some embodiments, the polypeptide comprises a firstsequence of at least 8 or 9 contiguous amino acids with at least 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68% 69%, 70%, 71%, 72%, 73% 74%, 75%,76% 77% 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to acorresponding wild-type sequence; and a second sequence of at least 16or 17 contiguous amino acids with at least 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73% 74% 75%, 76%, 77% 78% 79%, 80%,81%, 82%, 83%, 84% 85%, 86% 87%, 88% 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to a corresponding wild-typesequence.

In some embodiments, the second peptide is longer than the firstpeptide. In some embodiments, the first peptide is longer than thesecond peptide. In some embodiments, the first peptide has a length ofat least 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24;25; 26; 27; 28; 29; 30; 40; 50; 60; 70; 80; 90; 100; 150; 200; 250; 300;350; 400; 450; 500; 600; 700; 800; 900; 1,000; 1,500; 2,000; 2,500;3,000; 4,000; 5,000; 7,500; or 10,000 amino acids. In some embodiments,the second peptide has a length of at least 17; 18; 19; 20; 21; 22; 23;24; 25; 26; 27; 28; 29; 30; 40; 50; 60; 70; 80; 90; 100; 150; 200; 250;300; 350; 400; 450; 500; 600; 700; 800; 900; 1,000; 1,500; 2,000; 2,500;3,000; 4,000; 5,000; 7,500; or 10,000 amino acids. In some embodiments,the first peptide comprises a sequence of at least 9 contiguous aminoacids with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73% 74% 75%, 76%, 77% 78% 79%, 80%, 81%, 82%, 83%, 84%,85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to a corresponding wild-type sequence. In some embodiments,the second peptide comprises a sequence of at least 17 contiguous aminoacids with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75% 76% 77%, 78%, 79%, 80%, 81%, 82%, 83% 84%,85%, 86% 87% 88% 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity to a corresponding wild-type sequence.

In some embodiments, the first peptide, the second peptide or bothcomprise at least one flanking sequence, wherein the at least oneflanking sequence is upstream or downstream of the neoepitope. In someembodiments, the at least one flanking sequence has at least 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to a corresponding wild-type sequence. In some embodiments, theat least one flanking sequence comprises a non-wild-type sequence. Insome embodiments, the at least one flanking sequence is a N-terminusflanking sequence. In some embodiments, the at least one flankingsequence is a C-terminus flanking sequence. In some embodiments, the atleast one flanking sequence of the first peptide has at least 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to the at least one flanking sequence of the second peptide. Insome embodiments, the at least one flanking region of the first peptideis different from the at least one flanking region of the secondpeptide. In some embodiments, the at least one flanking residuecomprises the mutation.

In some embodiments, a peptide comprises a neoepitope sequencecomprising at least one mutant amino acid. In some embodiments, apeptide comprises a neoepitope sequence comprising at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30 or more mutant amino acids. In someembodiments, a peptide comprises a neoepitope sequence derived from aprotein comprising at least one mutant amino acid and at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more non-mutant amino acids. In someembodiments, a peptide comprises a neoepitope sequence derived from aprotein comprising at least one mutant amino acid and at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more non-mutant amino acids upstreamof the least one mutant amino acid. In some embodiments, a peptidecomprises a neoepitope sequence derived from a protein comprising atleast one mutant amino acid and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30 or more non-mutant amino acids downstream of the least one mutantamino acid. In some embodiments, a peptide comprises a neoepitopesequence derived from a protein comprising at least one mutant aminoacid; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or morenon-mutant amino acids upstream of the least one mutant amino acid; andat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutant aminoacids downstream of the least one mutant amino acid.

In some embodiments, a peptide comprises a neoantigenic peptide sequencedepicted in Tables 1 to 14. In some embodiments, a peptide comprises aneoepitope sequence depicted in Tables 1 to 14. In some embodiments, apeptide comprises a neoepitope sequence comprising at least one mutantamino acid (underlined amino acid) as depicted in Tables 1 to 14. Insome embodiments, a peptide comprises a neoepitope sequence comprisingat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more mutant aminoacids (underlined amino acids) as depicted in Tables 1 to 14. In someembodiments, a peptide comprises a neoepitope sequence comprising atleast one mutant amino acid (underlined amino acid) and at least onebolded amino acid as depicted in Tables 1 to 14. In some embodiments, apeptide comprises a neoepitope sequence derived from a proteincomprising at least one mutant amino acid (underlined amino acid) and atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutant aminoacids as depicted in Tables 1 to 14. In some embodiments, a peptidecomprises a neoepitope sequence derived from a protein comprising atleast one mutant amino acid (underlined amino acid) and at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more non-mutant amino acids upstreamof the least one mutant amino acid as depicted in Tables 1 to 14. Insome embodiments, a peptide comprises a neoepitope sequence derived froma protein comprising at least one mutant amino acid (underlined aminoacid) and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or morenon-mutant amino acids downstream of the least one mutant amino acid asdepicted in Tables 1 to 14. In some embodiments, a peptide comprises aneoepitope sequence derived from a protein comprising at least onemutant amino acid (underlined amino acid), at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30 or more non-mutant amino acids upstream of the leastone mutant amino acid, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30 or more non-mutant amino acids downstream of the least one mutantamino acid as depicted in Tables 1 to 14.

In some embodiments, a peptide comprises a neoepitope sequence derivedfrom a protein comprising at least one mutant amino acid and a sequenceupstream of the least one mutant amino acid with at least 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity toa corresponding wild type sequence. In some embodiments, a peptidecomprises a neoepitope sequence derived from a protein comprising atleast one mutant amino acid and a sequence downstream of the least onemutant amino acid with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to a corresponding wildtype sequence. In some embodiments, a peptide comprises a neoepitopesequence derived from a protein comprising at least one mutant aminoacid, a sequence upstream of the least one mutant amino acid with atleast 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to a corresponding wild type sequence, and a sequencedownstream of the least one mutant amino acid with at least 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to a corresponding wild type sequence.

In some embodiments, a peptide comprises a neoepitope sequence derivedfrom a protein comprising at least one mutant amino acid and a sequenceupstream of the least one mutant amino acid comprising least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids with at least60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to a corresponding wild type sequence. In someembodiments, a peptide comprises a neoepitope sequence derived from aprotein comprising at least one mutant amino acid and a sequencedownstream of the least one mutant amino acid comprising least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids with atleast 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to a corresponding wild type sequence. In someembodiments, a peptide comprises a neoepitope sequence derived from aprotein comprising at least one mutant amino acid, a sequence upstreamof the least one mutant amino acid comprising least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30 or more contiguous amino acids with at least 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to a corresponding wild type sequence, and a sequencedownstream of the least one mutant amino acid comprising least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids with atleast 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,8′7%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to a corresponding wild type sequence.

In some embodiments, a peptide comprises a neoepitope sequence derivedfrom a protein comprising at least one mutant amino acid (underlinedamino acid) as depicted in Tables 1 to 14 and a sequence upstream of theleast one mutant amino acid with at least 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to acorresponding wild type sequence. In some embodiments, a peptidecomprises a neoepitope sequence derived from a protein comprising atleast one mutant amino acid (underlined amino acid) as depicted inTables 1 to 14 and a sequence downstream of the least one mutant aminoacid with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a corresponding wild typesequence. In some embodiments, a peptide comprises a neoepitope sequencederived from a protein comprising at least one mutant amino acid(underlined amino acid) as depicted in Tables 1 to 14, a sequenceupstream of the least one mutant amino acid with at least 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity toa corresponding wild type sequence, and a sequence downstream of theleast one mutant amino acid with at least 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to acorresponding wild type sequence.

In some embodiments, a peptide comprises a neoepitope sequence derivedfrom a protein comprising at least one mutant amino acid (underlinedamino acid) as depicted in Tables 1 to 14 and a sequence upstream of theleast one mutant amino acid comprising least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30 or more contiguous amino acids with at least 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity toa corresponding wild type sequence. In some embodiments, a peptidecomprises a neoepitope sequence derived from a protein comprising atleast one mutant amino acid (underlined amino acid) as depicted inTables 1 to 14 and a sequence downstream of the least one mutant aminoacid comprising least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or morecontiguous amino acids with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% sequence identity to a correspondingwild type sequence. In some embodiments, a peptide comprises aneoepitope sequence derived from a protein comprising at least onemutant amino acid (underlined amino acid) as depicted in Tables 1 to 14,a sequence upstream of the least one mutant amino acid comprising least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acidswith at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% sequence identity to a corresponding wild type sequence, anda sequence downstream of the least one mutant amino acid comprisingleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous aminoacids with at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a corresponding wild typesequence.

In some embodiments, a peptide comprising a KRAS G12C mutation comprisesa sequence of MTEYKLVVVGACGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQE. In some embodiments, a peptide comprising a KRAS G12 Cmutation comprises a neoepitope sequence of KLVVVGACGV. In someembodiments, a peptide comprising a KRAS G12 C mutation comprises aneoepitope sequence of LVVVGACGV. In some embodiments, a peptidecomprising a KRAS G12 C mutation comprises a neoepitope sequence ofVVGACGVGK. In some embodiments, a peptide comprising a KRAS G12 Cmutation comprises a neoepitope sequence of VVVGACGVGK.

In some embodiments, a peptide comprising a KRAS G12D mutation comprisesa sequence ofMTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQE. In someembodiments, a peptide comprising a KRAS G12D mutation comprises aneoepitope sequence of VVGADGVGK. In some embodiments, a peptidecomprising a KRAS G12D mutation comprises a neoepitope sequence ofVVVGADGVGK. In some embodiments, a peptide comprising a KRAS G12Dmutation comprises a neoepitope sequence of KLVVVGADGV. In someembodiments, a peptide comprising a KRAS G12D mutation comprises aneoepitope sequence of LVVVGADGV.

In some embodiments, a peptide comprising a KRAS G12V mutation comprisesa sequence ofMTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQE. In someembodiments, a peptide comprising a KRAS G12V mutation comprises aneoepitope sequence of KLVVVGAVGV. In some embodiments, a peptidecomprising a KRAS G12V mutation comprises a neoepitope sequence ofLVVVGAVGV. In some embodiments, a peptide comprising a KRAS G12Vmutation comprises a neoepitope sequence of VVGAVGVGK. In someembodiments, a peptide comprising a KRAS G12V mutation comprises aneoepitope sequence of VVVGAVGVGK.

In some embodiments, a peptide comprising a KRAS Q61H mutation comprisesa sequence ofAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGHEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPM. In some embodiments, a peptidecomprising a KRAS Q61H mutation comprises a neoepitope sequence ofILDTAGHEEY.

In some embodiments, a peptide comprising a KRAS Q61L mutation comprisesa sequence ofAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGLEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPM. In some embodiments, a peptidecomprising a KRAS Q61L mutation comprises a neoepitope sequence ofILDTAGLEEY. In some embodiments, a peptide comprising a KRAS Q61Lmutation comprises a neoepitope sequence of LLDILDTAGL.

In some embodiments, a peptide comprising a NRAS Q61K mutation comprisesa sequence ofAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGKEEYSAMRDQYMRTGEGFLCVFAINNSKSFADINLYREQIKRVKDSDDVPM. In some embodiments, a peptidecomprising a NRAS Q61K mutation comprises a neoepitope sequence ofILDTAGKEEY.

In some embodiments, a peptide comprising a NRAS Q61R mutation comprisesa sequence ofAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGREEYSAMRDQYMRTGEGFLCVFAINNSKSFADINLYREQIKRVKDSDDVPM. In some embodiments, a peptidecomprising a NRAS Q61R mutation comprises a neoepitope sequence ofILDTAGREEY.

In some embodiments, a peptide comprising a mutation of a TMPRSS2:ERGfusion comprises a neoepitope sequence ofMALNS::EALSVVSEDQSLFECAYGTPHLAKTEMTASSSSDYGQTSKMSPRVPQQDWALNSEALS V. Insome embodiments, a peptide comprising a mutation of a TMPRSS2:ERGfusion comprises a neoepitope sequence of ALNSEALSVV. In someembodiments, a peptide comprising a mutation of a TMPRSS2:ERG fusioncomprises a neoepitope sequence of MALNSEALSV. In some embodiments, apeptide comprising a RAS Q61H mutation comprises a sequence ofTCLLDILDTAGHEEYSAMRDQYM.

In some embodiments, a peptide comprising a RAS Q61H mutation comprisesa sequence provided in Table 3. In some embodiments, a peptide sequenceprovided in Table 3 binds to or is predicted to bind to a proteinencoded by an HLA allele, which allele is provided in a correspondingcolumn in Table 3 next to the peptide sequence.

TABLE 3 Peptide Sequences Comprising RAS Q61HMutation, Corresponding HLA Allele, and Rank of Binding PotentialRank of Binding Peptide Allele Potential ILDTAGHEEY HLA-A36:01 1ILDTAGHEEY HLA-A01:01 2 DTAGHEEYSAM HLA-A26:01 3 DTAGHEEYSAM HLA-A25:014 GHEEYSAM HLA-B15:09 4 DTAGHEEY HLA-A26:01 5 ILDTAGHEE HLA-C08:02 5AGHEEYSAM HLA-C01:02 6 AGHEEYSAM HLA-B46:01 6 DTAGHEEY HLA-A25:01 6DTAGHEEY HLA-A01:01 6 DTAGHEEY HLA-B18:01 7 DTAGHEEY HLA-A36:01 7ILDTAGHEE HLA-C05:01 7 ILDTAGHEE HLA-A02:07 7 ILDTAGHEEY HLA-A29:02 7ILDTAGHEEY HLA-C08:02 7 HEEYSAMRD HLA-B49:01 8 TAGHEEYSA HLA-B35:03 8DTAGHEEYS HLA-A68:02 9 DTAGHEEYSAMR HLA-A68:01 9 GHEEYSAM HLA-B39:01 9ILDTAGHEE HLA-A01:01 9 LDTAGHEEY HLA-B53:01 9 HEEYSAMRD HLA-B41:01 10ILDTAGHEE HLA-A36:01 10 DTAGHEEY HLA-B58:01 11 LLDILDTAGH HLA-A01:01 12TAGHEEYSAM HLA-B35:03 12 LDTAGHEEY HLA-B35:01 13 DILDTAGHE HLA-A26:01 14DTAGHEEY HLA-C12:03 14 ILDTAGHEEY HLA-C05:01 14 AGHEEYSAM HLA-A30:02 15DILDTAGHEEY HLA-A25:01 15 DTAGHEEY HLA-C02:02 15 ILDTAGHEE HLA-C04:01 15DILDTAGH HLA-A26:01 16 ILDTAGHEE HLA-A02:01 16 LDTAGHEEY HLA-A29:02 16ILDTAGHE HLA-A01:01 17 LDTAGHEEY HLA-B18:01 17 AGHEEYSAM HLA-C14:03 18DILDTAGHEEY HLA-A29:02 18 DTAGHEEYS HLA-A26:01 18 ILDTAGHEEY HLA-B15:0118 DTAGHEEYSA HLA-A68:02 19 ILDTAGHE HLA-C05:01 19 ILDTAGHEEY HLA-A02:0719 ILDTAGHEEY HLA-A30:02 19 LDTAGHEEY HLA-A36:01 19 AGHEEYSAM HLA-C14:0220 AGHEEYSAM HLA-B15:03 20 LLDILDTAGH HLA-A02:07 20

In some embodiments, a peptide comprising a RAS Q61R mutation comprisesa sequence of TCLLDILDTAGREEYSAMRDQYM. In some embodiments, a peptidecomprising a RAS Q61R mutation comprises a sequence provided in Table 4.In some embodiments, a peptide sequence provided in Table 4 binds to oris predicted to bind to a protein encoded by an HLA allele, which alleleis provided in a corresponding column in Table 4 next to the peptidesequence.

TABLE 4 Peptide Sequences Comprising RAS Q61R Mutation,Corresponding HLA Allele, and Rank of Binding Potential Rank of PeptideAllele Binding Potential ILDTAGREEY HLA-A36:01 1 ILDTAGREEY HLA-A01:01 2DTAGREEYSAM HLA-A26:01 3 DILDTAGR HLA-A33:03 4 DILDTAGR HLA-A68:01 5DTAGREEY HLA-A26:01 6 DTAGREEYSAM HLA-A25:01 6 CLLDILDTAGR HLA-A74:01 7DTAGREEY HLA-A01:01 7 REEYSAMRD HLA-B41:01 7 GREEYSAMR HLA-B27:05 8ILDTAGREE HLA-C08:02 8 ILDTAGREEY HLA-A29:02 8 REEYSAMRD HLA-B49:01 8AGREEYSAM HLA-B46:01 9 DTAGREEY HLA-B18:01 9 DTAGREEY HLA-A25:01 9DTAGREEY HLA-A36:01 9 DILDTAGR HLA-A74:01 10 DILDTAGRE HLA-A26:01 10ILDTAGREE HLA-C05:01 10 DILDTAGR HLA-A26:01 11 GREEYSAM HLA-B39:01 11AGREEYSAM HLA-B15:03 12 GREEYSAM HLA-C07:02 12 ILDTAGREE HLA-A01:01 12TAGREEYSA HLA-B35:03 12 ILDTAGREEY HLA-A30:02 13 DTAGREEYS HLA-A68:02 14ILDTAGRE HLA-A01:01 14 CLLDILDTAGR HLA-A31:01 15 DTAGREEYSAMR HLA-A68:0115 LLDILDTAGR HLA-A01:01 15 DTAGREEY HLA-B58:01 16 ILDTAGREEY HLA-C08:0216 DILDTAGR HLA-A31:01 17 ILDTAGREE HLA-C04:01 17 ILDTAGREEY HLA-A32:0117 LLDILDTAGR HLA-A74:01 17 TAGREEYSAM HLA-B35:03 17 DILDTAGREEYHLA-A32:01 18 ILDTAGRE HLA-C05:01 18 ILDTAGREE HLA-A02:07 18 REEYSAMRDHLA-B40:01 18 AGREEYSAM HLA-B15:01 19 AGREEYSAMR HLA-A31:01 19 ILDTAGREHLA-A36:01 19 LDILDTAGR HLA-A68:01 19 LDTAGREEY HLA-A29:02 19 LDTAGREEYHLA-B35:01 19 REEYSAMRD HLA-B45:01 19 REEYSAMRDQY HLA-A36:01 19 DTAGREEYHLA-C02:02 20

In some embodiments, a peptide comprising a RAS Q61K mutation comprisesa sequence of TCLLDILDTAGKEEYSAMRDQYM. In some embodiments, a peptidecomprising a RAS Q61K mutation comprises a sequence provided in Table 5.In some embodiments, a peptide sequence provided in Table 5 binds to oris predicted to bind to a protein encoded by an HLA allele, which alleleis provided in a corresponding column in Table 5 next to the peptidesequence.

TABLE 5 Peptide Sequences Comprising RAS Q61K Mutation,Corresponding HLA Allele, and Rank of Potential Binding Rank of PeptideAllele Binding Potential ILDTAGKEEY HLA-A36:01 1 ILDTAGKEEY HLA-A01:01 2DTAGKEEYSAM HLA-A26:01 3 CLLDILDTAGK HLA-A03:01 4 DTAGKEEY HLA-A01:01 5DTAGKEEY HLA-A26:01 5 DTAGKEEYSAM HLA-A25:01 5 AGKEEYSAM HLA-B46:01 6DILDTAGKE HLA-A26:01 7 KEEYSAMRD HLA-B41:01 7 DTAGKEEY HLA-B18:01 8GKEEYSAM HLA-B15:03 8 ILDTAGKEE HLA-C08:02 8 ILDTAGKEEY HLA-A29:02 8DTAGKEEYS HLA-A68:02 9 LDTAGKEEY HLA-B53:01 9 TAGKEEYSA HLA-B35:03 9DILDTAGK HLA-A68:01 10 DTAGKEEY HLA-A36:01 10 KEEYSAMRD HLA-B49:01 10LDTAGKEEY HLA-C07:01 10 DTAGKEEYSAMR HLA-A68:01 11 ILDTAGKEE HLA-C05:0111 ILDTAGKEEY HLA-C08:02 11 LLDILDTAGK HLA-A01:01 12 AGKEEYSAMHLA-A30:02 13 DTAGKEEY HLA-A25:01 13 DTAGKEEYS HLA-A26:01 13 ILDTAGKEHLA-C05:01 13 LDTAGKEEY HLA-B35:01 13 AGKEEYSAMR HLA-A31:01 14 DILDTAGKHLA-A33:03 14 ILDTAGKE HLA-A01:01 14 ILDTAGKEE HLA-A01:01 14 ILDTAGKEEHLA-A02:07 14 TAGKEEYSAM HLA-B35:03 14 AGKEEYSAM HLA-B15:01 15ILDTAGKEEY HLA-A30:02 15 LDTAGKEEY HLA-B46:01 15 DTAGKEEY HLA-B58:01 16ILDTAGKEEY HLA-C05:01 17 AGKEEYSAM HLA-A30:01 18 AGKEEYSAM HLA-B15:03 18DTAGKEEY HLA-C02:02 18 LDTAGKEEY HLA-A29:02 18

In some embodiments, a peptide comprising a RAS Q61L mutation comprisesa sequence of TCLLDILDTAGLEEYSAMRDQYM. In some embodiments, a peptidecomprising a RAS Q61L mutation comprises a sequence provided in Table 6.In some embodiments, a peptide sequence provided in Table 6 binds to oris predicted to bind to a protein encoded by an HLA allele, which alleleis provided in a corresponding column in Table 6 next to the peptidesequence.

TABLE 6 Peptide Sequences Comprising RAS Q61LMutation, Corresponding HLA Allele,  and Rank of Binding PotentialRank of Binding Peptide Allele Potential ILDTAGLEEY HLA-A36:01 1ILDTAGLEEY HLA-A01:01 2 LLDILDTAGL HLA-A02:07 3 GLEEYSAMRDQY HLA-A36:014 DTAGLEEY HLA-A25:01 5 DTAGLEEY HLA-A26:01 5 DTAGLEEYSAM HLA-A26:01 5DTAGLEEY HLA-A01:01 6 ILDTAGLEE HLA-C08:02 6 ILDTAGLEE HLA-A01:01 6CLLDILDTAGL HLA-A02:04 7 ILDTAGLEE HLA-A36:01 7 LLDILDTAGL HLA-A01:01 7DILDTAGL HLA-B14:02 8 DILDTAGLEEY HLA-A25:01 8 DTAGLEEYS HLA-A68:02 8DTAGLEEYSAM HLA-A25:01 8 GLEEYSAMR HLA-A74:01 8 ILDTAGLE HLA-A01:01 8DILDTAGLEEY HLA-A26:01 9 DTAGLEEY HLA-A36:01 9 ILDTAGLEEY HLA-A29:02 9DILDTAGL HLA-B08:01 10 DTAGLEEY HLA-B18:01 10 ILDTAGLEE HLA-A02:07 10LDTAGLEEY HLA-B35:01 10 CLLDILDTAGL HLA-A02:01 11 DTAGLEEY HLA-C02:02 11ILDTAGLEE HLA-C05:01 11 ILDTAGLEEY HLA-C08:02 11 ILDTAGLEEY HLA-A02:0711 LLDILDTAGL HLA-C08:02 11 DILDTAGL HLA-A26:01 12 LDTAGLEEY HLA-B53:0112 DTAGLEEY HLA-C03:02 13 DTAGLEEY HLA-B58:01 13 ILDTAGLEEY HLA-A30:0213 LLDILDTAGL HLA-C05:01 13 LLDILDTAGL HLA-C04:01 13 DTAGLEEYSAMRHLA-A68:01 14 ILDTAGLE HLA-A36:01 15 LLDILDTAGL HLA-A02:01 15 AGLEEYSAMHLA-B15:03 16 DTAGLEEYSA HLA-A68:02 16 GLEEYSAMRDQY HLA-A01:01 16ILDTAGLE HLA-C04:01 16 ILDTAGLEEY HLA-B15:01 16 LDILDTAGL HLA-B37:01 16AGLEEYSAM HLA-A30:02 17 AGLEEYSAM HLA-B48:01 17 AGLEEYSAMR HLA-A31:01 17ILDTAGLEE HLA-C04:01 17 LDTAGLEEY HLA-C03:02 17 AGLEEYSAM HLA-C14:02 18GLEEYSAMR HLA-A31:01 18 LEEYSAMRD HLA-B41:01 18 LLDILDTAGLE HLA-A01:0118 AGLEEYSAM HLA-C14:03 19 LDILDTAGL HLA-B40:02 19 LDTAGLEEY HLA-A29:0219 DILDTAGLE HLA-A26:01 20 DTAGLEEY HLA-B15:01 20 ILDTAGLEEY HLA-A02:0120 LDTAGLEEY HLA-A36:01 20 LDTAGLEEY HLA-B46:01 20 DTAGLEEY HLA-A68:0221 DTAGLEEY HLA-C12:03 21 ILDTAGLE HLA-C05:01 21 LDTAGLEEY HLA-B18:01 21LEEYSAMRD HLA-B49:01 21 TAGLEEYSA HLA-B54:01 21 DILDTAGLEEY HLA-A29:0222 GLEEYSAM HLA-C05:01 22

In some embodiments, a peptide comprising a RAS G12A mutation comprisesa sequence of MTEYKLVVVGAAGVGKSALTIQL. In some embodiments, a peptidecomprising a RAS G12A mutation comprises a sequence provided in Table 7.In some embodiments, a peptide sequence provided in Table 7 binds to oris predicted to bind to a protein encoded by an HLA allele, which alleleis provided in a corresponding column in Table 7 next to the peptidesequence.

TABLE 7 Peptide Sequences Comprising RAS G12AMutation, Corresponding HLA Allele, and Rank of Binding PotentialRank of Binding Peptide Allele Potential AAGVGKSAL HLA-C03:04 1VVVGAAGVGK HLA-A11:01 1 VVGAAGVGK HLA-A11:01 2 TEYKLVVVGAA HLA-B50:01 3VVGAAGVGK HLA-A03:01 3 VVVGAAGVGK HLA-A68:01 3 AAGVGKSAL HLA-C08:02 4AAGVGKSAL HLA-C08:01 4 AAGVGKSAL HLA-B46:01 4 AAGVGKSAL HLA-B81:01 5GAAGVGKSAL HLA-B48:01 5 LVVVGAAGV HLA-A68:02 5 AAGVGKSAL HLA-C03:04 1VVVGAAGVGK HLA-A11:01 1 VVGAAGVGK HLA-A11:01 2 TEYKLVVVGAA HLA-B50:01 3VVGAAGVGK HLA-A03:01 3 VVVGAAGVGK HLA-A68:01 3 AAGVGKSAL HLA-C08:02 4AAGVGKSAL HLA-C08:01 4 AAGVGKSAL HLA-B46:01 4 AAGVGKSAL HLA-B81:01 5AAGVGKSAL HLA-C03:02 5 AAGVGKSAL HLA-C01:02 5 GAAGVGKSAL HLA-B48:01 5LVVVGAAGV HLA-A68:02 5 AAGVGKSAL HLA-C03:03 6 VVGAAGVGK HLA-A68:01 6GAAGVGKSAL HLA-B81:01 7 VVVGAAGVGK HLA-A03:01 7 AAGVGKSAL HLA-C05:01 8AAGVGKSAL HLA-C12:03 8 GAAGVGKSA HLA-B46:01 8 VVGAAGVGK HLA-A30:01 8GAAGVGKSA HLA-B55:01 9 KLVVVGAAGV HLA-A02:01 9 AGVGKSAL HLA-B08:01 10GAAGVGKSAL HLA-C03:04 10 AAGVGKSAL HLA-C17:01 11 GAAGVGKSAL HLA-C03:0311 VVVGAAGV HLA-A68:02 11 YKLVVVGAA HLA-B54:01 11 AAGVGKSAL HLA-B48:0112 AGVGKSAL HLA-C03:04 12 AGVGKSAL HLA-C07:01 12 VVVGAAGVGK HLA-A30:0112 AAGVGKSA HLA-B46:01 13 KLVVVGAAGV HLA-A02:07 13 YKLVVVGAA HLA-B50:0113 AAGVGKSAL HLA-B07:02 14 GAAGVGKSAL HLA-A68:02 14 VVGAAGVGK HLA-A74:0114 AGVGKSAL HLA-C08:01 15 GAAGVGKSAL HLA-C17:01 15 GAAGVGKSAL HLA-C08:0116 GAAGVGKSAL HLA-B35:03 16 AAGVGKSAL HLA-C02:02 17 AAGVGKSAL HLA-B35:0317 AAGVGKSAL HLA-C12:02 17 AAGVGKSAL HLA-C14:03 17 GAAGVGKSA HLA-B50:0117 AGVGKSAL HLA-C03:02 18 GAAGVGKSA HLA-C03:04 18 LVVVGAAGV HLA-B55:0118 TEYKLVVVGAA HLA-B41:01 18 AGVGKSAL HLA-C01:02 19 GAAGVGKSA HLA-B54:0119 GAAGVGKSAL HLA-B07:02 19 VGAAGVGKSA HLA-B55:01 19 AGVGKSAL HLA-B48:0120 AGVGKSALTI HLA-B49:01 20 VVVGAAGV HLA-B55:01 20

In some embodiments, a peptide comprising a RAS G12C mutation comprisesa sequence of MTEYKLVVVGACGVGKSALTIQL. In some embodiments, a peptidecomprising a RAS G12C mutation comprises a sequence provided in Table 8.In some embodiments, a peptide sequence provided in Table 8 binds to oris predicted to bind to a protein encoded by an HLA allele, which alleleis provided in a corresponding column in Table 8 next to the peptidesequence.

TABLE 8 Peptide Sequences Comprising RAS G12CMutation, Corresponding HLA Allele, and Rank of Binding PotentialRank of Binding Peptide Allele Potential VVVGACGVGK HLA-A11:01 1VVGACGVGK HLA-A03:01 2 VVGACGVGK HLA-A11:01 3 VVVGACGVGK HLA-A68:01 4VVGACGVGK HLA-A68:01 5 VVVGACGVGK HLA-A03:01 5 VVGACGVGK HLA-A30:01 6ACGVGKSAL HLA-B81:01 7 ACGVGKSAL HLA-C01:02 7 ACGVGKSAL HLA-C14:03 8ACGVGKSAL HLA-C03:04 9 VVVGACGVGK HLA-A30:01 9 ACGVGKSAL HLA-C14:02 10CGVGKSAL HLA-B08:01 10 KLVVVGACGV HLA-A02:01 10 ACGVGKSAL HLA-B07:02 11GACGVGKSAL HLA-B48:01 12 GACGVGKSAL HLA-C03:03 13 ACGVGKSAL HLA-B48:0114 ACGVGKSAL HLA-B40:01 14 YKLVVVGAC HLA-B48:01 14 YKLVVVGAC HLA-B15:0314 GACGVGKSA HLA-B46:01 15 GACGVGKSAL HLA-C03:04 15 GACGVGKSALHLA-C01:02 15 LVVVGACGV HLA-A68:02 15 CGVGKSAL HLA-C03:04 16 GACGVGKSALHLA-C08:02 16 VVGACGVGK HLA-A74:01 16

In some embodiments, a peptide comprising a RAS G12D mutation comprisesa sequence of MTEYKLVVVGADGVGKSALTIQL. In some embodiments, a peptidecomprising a RAS G12D mutation comprises a sequence provided in Table 9.In some embodiments, a peptide sequence provided in Table 9 binds to oris predicted to bind to a protein encoded by an HLA allele, which alleleis provided in a corresponding column in Table 9 next to the peptidesequence

TABLE 9 Peptide Sequences Comprising RAS G12DMutation, Corresponding HLA Allele, and Rank of Binding PotentialRank of Binding Peptide Allele Potential GADGVGKSAL HLA-C08:02 1GADGVGKSAL HLA-C05:01 2 VVVGADGVGK HLA-A11:01 3 DGVGKSAL HLA-B14:02 4VVGADGVGK HLA-A11:01 4 VVGADGVGK HLA-A03:01 5 DGVGKSAL HLA-B08:01 6VVVGADGVGK HLA-A68:01 6 GADGVGKSAL HLA-C03:03 7 VVGADGVGK HLA-A30:01 7ADGVGKSAL HLA-B37:01 8 GADGVGKSAL HLA-C08:01 8 VVGADGVGK HLA-A68:01 8GADGVGKSA HLA-C08:02 9 GADGVGKSAL HLA-B35:03 9 GADGVGKS HLA-C05:01 10GADGVGKSA HLA-C05:01 10 ADGVGKSAL HLA-C07:01 11 VVVGADGVGK HLA-A03:01 11ADGVGKSAL HLA-B40:02 12 ADGVGKSAL HLA-B46:01 13 GADGVGKSAL HLA-C03:04 13ADGVGKSAL HLA-B81:01 14 GADGVGKSAL HLA-C17:01 14 VVVGADGVGK HLA-A30:0114 GADGVGKSA HLA-B35:03 15 GADGVGKSA HLA-B46:01 15 GADGVGKSAL HLA-B48:0115 KLVVVGADGV HLA-A02:01 15 LVVVGADGV HLA-A68:02 15 VGADGVGKSAHLA-B55:01 15 VVGADGVGK HLA-A74:01 16 GADGVGKSA HLA-B53:01 17 KLVVVGADGVHLA-A02:07 17 VGADGVGK HLA-A68:01 17 YKLVVVGAD HLA-B48:01 17 ADGVGKSALHLA-C14:03 18 DGVGKSALTI HLA-B51:01 18 VGADGVGK HLA-A11:01 18

In some embodiments, a peptide comprising a RAS G12R mutation comprisesa sequence of MTEYKLVVVGARGVGKSALTIQL. In some embodiments, a peptidecomprising a RAS G12R mutation comprises a sequence provided in Table10. In some embodiments, a peptide sequence provided in Table 10 bindsto or is predicted to bind to a protein encoded by an HLA allele, whichallele is provided in a corresponding column in Table 10 next to thepeptide sequence.

TABLE 10 Peptide Sequences Comprising RAS G12RMutation, Corresponding HLA Allele, and Rank of Binding PotentialRank of Binding Peptide Allele Potential VVGARGVGK HLA-A11:01 1VVVGARGVGK HLA-A68:01 1 GARGVGKSA HLA-B46:01 2 ARGVGKSAL HLA-B27:05 3GARGVGKSA HLA-B55:01 3 RGVGKSAL HLA-C07:01 4 VVGARGVGK HLA-A30:01 5ARGVGKSAL HLA-B38:01 6 ARGVGKSAL HLA-B14:02 6 VVGARGVGK HLA-A68:01 6VVVGARGVGK HLA-A03:01 7 GARGVGKSAL HLA-B48:01 8 RGVGKSAL HLA-B48:01 8RGVGKSALTI HLA-A23:01 8 ARGVGKSAL HLA-C06:02 9 GARGVGKSA HLA-A30:01 9GARGVGKSAL HLA-B81:01 9 VVVGARGVGK HLA-A30:01 9 GARGVGKSAL HLA-B07:02 10LVVVGARGV HLA-C06:02 10 RGVGKSAL HLA-B81:01 10 VVGARGVGK HLA-A74:01 11KLVVVGARGV HLA-A02:01 12 LVVVGARGV HLA-B55:01 12 YKLVVVGAR HLA-A33:03 12KLVVVGAR HLA-A74:01 13 KLVVVGARGV HLA-B13:02 13 RGVGKSAL HLA-C01:02 13LVVVGARGV HLA-A68:02 14 VVVGARGV HLA-B55:01 14 ARGVGKSAL HLA-B15:09 15ARGVGKSAL HLA-C14:03 16 GARGVGKSA HLA-B54:01 16 VVVGARGV HLA-B52:01 16

In some embodiments, a peptide comprising a RAS G12S mutation comprisesa sequence of MTEYKLVVVGASGVGKSALTIQL. In some embodiments, a peptidecomprising a RAS G12S mutation comprises a sequence provided in Table11. In some embodiments, a peptide sequence provided in Table 11 bindsto or is predicted to bind to a protein encoded by an HLA allele, whichallele is provided in a corresponding column in Table 11 next to thepeptide sequence.

TABLE 11 Peptide Sequences Comprising RAS G12SMutation, Corresponding HLA Allele, and Rank of Binding PotentialRank of Binding Peptide Allele Potential VVVGASGVGK HLA-A11:01 1VVGASGVGK HLA-A11:01 2 VVGASGVGK HLA-A03:01 3 VVVGASGVGK HLA-A68:01 4ASGVGKSAL HLA-C03:04 5 ASGVGKSAL HLA-B46:01 5 VVGASGVGK HLA-A68:01 6VVVGASGVGK HLA-A03:01 6 ASGVGKSAL HLA-C01:02 7 GASGVGKSAL HLA-B48:01 7ASGVGKSAL HLA-C07:01 8 ASGVGKSAL HLA-C08:02 9 GASGVGKSAL HLA-B81:01 9SGVGKSAL HLA-B08:01 9 ASGVGKSAL HLA-C03:03 10 ASGVGKSAL HLA-C03:02 10SGVGKSAL HLA-B14:02 10 VVGASGVGK HLA-A30:01 10 ASGVGKSAL HLA-C08:01 11VVVGASGVGK HLA-A30:01 11 GASGVGKSAL HLA-B35:03 12 SGVGKSAL HLA-C07:01 12ASGVGKSAL HLA-B81:01 13 GASGVGKSA HLA-B55:01 13 GASGVGKSAL HLA-C03:03 13KLVVVGASGV HLA-A02:01 13 LVVVGASGV HLA-A68:02 13 SGVGKSAL HLA-C01:02 13ASGVGKSA HLA-B46:01 14 ASGVGKSAL HLA-C15:02 14 GASGVGKSAL HLA-C08:01 15SGVGKSAL HLA-C03:04 15 ASGVGKSAL HLA-C05:01 16 GASGVGKSAL HLA-C03:04 16VVGASGVGK HLA-A74:01 16 ASGVGKSAL HLA-B48:01 17 GASGVGKSAL HLA-C01:02 17SGVGKSAL HLA-C03:02 17 SGVGKSALTI HLA-A23:01 17 VGASGVGKSA HLA-B55:01 18ASGVGKSAL HLA-C12:03 19 ASGVGKSAL HLA-B57:03 19 KLVVVGASGV HLA-A02:07 19SGVGKSAL HLA-B81:01 19 ASGVGKSAL HLA-C17:01 20 KLVVVGASG HLA-A32:01 20

In some embodiments, a peptide comprising a RAS G12V mutation comprisesa sequence of MTEYKLVVVGAVGVGKSALTIQL. In some embodiments, a peptidecomprising a RAS G12V mutation comprises a sequence provided in Table12. In some embodiments, a peptide sequence provided in Table 12 bindsto or is predicted to bind to a protein encoded by an HLA allele, whichallele is provided in a corresponding column in Table 12 next to thepeptide sequence.

TABLE 12 Peptide Sequences Comprising RAS G12VMutation, Corresponding HLA Allele, and Rank of Binding PotentialRank of Binding Peptide Allele Potential VVGAVGVGK HLA-A03:01 1VVGAVGVGK HLA-A11:01 2 VVVGAVGVGK HLA-A11:01 2 VVVGAVGVGK HLA-A68:01 3VVGAVGVGK HLA-A68:01 4 LVVVGAVGV HLA-A68:02 5 VVGAVGVGK HLA-A30:01 5AVGVGKSAL HLA-B81:01 6 KLVVVGAVGV HLA-A02:01 6 AVGVGKSAL HLA-B46:01 7GAVGVGKSAL HLA-C03:03 7 GAVGVGKSAL HLA-B48:01 7 VVVGAVGVGK HLA-A03:01 7AVGVGKSAL HLA-C03:04 8 GAVGVGKSAL HLA-C03:04 8 KLVVVGAVGV HLA-A02:07 9VGVGKSAL HLA-B08:01 9 VVVGAVGV HLA-A68:02 9 AVGVGKSAL HLA-C08:02 10AVGVGKSAL HLA-B07:02 10 GAVGVGKSAL HLA-B35:03 10 AVGVGKSAL HLA-C08:01 11AVGVGKSAL HLA-C01:02 11 GAVGVGKSA HLA-B55:01 11 GAVGVGKSAL HLA-B81:01 11GAVGVGKSAL HLA-C08:01 11 KLVVVGAVGV HLA-B13:02 11 VGVGKSAL HLA-C03:04 11AVGVGKSAL HLA-A32:01 12 GAVGVGKSA HLA-B46:01 12 VGVGKSAL HLA-C03:02 12VGVGKSALTI HLA-A23:01 12 GAVGVGKSA HLA-B54:01 13 VGVGKSAL HLA-C01:02 .3AVGVGKSAL HLA-B48:01 14 AVGVGKSAL HLA-C03:03 14 AVGVGKSAL HLA-B42:01 14LVVVGAVGV HLA-B55:01 14 VGVGKSAL HLA-C08:01 14 VVGAVGVGK HLA-A74:01 14AVGVGKSAL HLA-C05:01 15 AVGVGKSAL HLA-C03:02 15 GAVGVGKSA HLA-C03:04 15KLVVVGAVGV HLA-A02:04 15 LVVVGAVGV HLA-A02:07 15 VGVGKSAL HLA-B14:02 15VVVGAVGVGK HLA-A30:01 15 VVGAVGVGK HLA-B81:01 16 VVVGAVGV HLA-B55:01 16AVGVGKSAL HLA-C14:03 17 AVGVGKSAL HLA-B15:01 17 LVVVGAVGV HLA-B54:01 17AVGVGKSA HLA-B55:01 18 AVGVGKSAL HLA-C17:01 18 GAVGVGKSA HLA-B50:01 19GAVGVGKSAL HLA-C17:01 19 YKLVVVGAV HLA-A02:04 19 GAVGVGKSAL HLA-B35:0120 VVGAVGVGK HLA-A31:01 20 YKLVVVGAV HLA-B51:01 20

In some embodiments, a peptide comprising a RAS G13C mutation comprisesa sequence of MTEYKLVVVGAGCVGKSALTIQL. In some embodiments, a peptidecomprising a RAS G13C mutation comprises a sequence provided in Table13. In some embodiments, a peptide sequence provided in Table 13 bindsto or is predicted to bind to a protein encoded by an HLA allele, whichallele is provided in a corresponding column in Table 13 next to thepeptide sequence.

TABLE 13 Peptide Sequences Comprising RAS G13CMutation, Corresponding HLA Allele, and Rank of Binding PotentialRank of Binding Peptide Allele Potential VVVGAGCVGK HLA-A11:01 1VVGAGCVGK HLA-A11:01 2 AGCVGKSAL HLA-C01:02 3 VVGAGCVGK HLA-A03:01 4VVVGAGCVGK HLA-A68:01 4 CVGKSALTI HLA-B13:02 5 VVGAGCVGK HLA-A68:01 5VVGAGCVGK HLA-A30:01 6 AGCVGKSAL HLA-B48:01 7 AGCVGKSAL HLA-C03:04 8GCVGKSALTI HLA-B49:01 8 AGCVGKSAL HLA-C08:02 9 VVVGAGCVGK HLA-A03:01 9KLVVVGAGC HLA-A30:02 10 GCVGKSAL HLA-C07:01 11 VVGAGCVGK HLA-A74:01 12AGCVGKSAL HLA-C14:03 13 KLVVVGAGC HLA-B15:01 14

In some embodiments, a peptide comprising a RAS G13D mutation comprisesa sequence of MTEYKLVVVGAGDVGKSALTIQL. In some embodiments, a peptidecomprising a RAS G13D mutation comprises a sequence provided in Table14. In some embodiments, a peptide sequence provided in Table 14 bindsto or is predicted to bind to a protein encoded by an HLA allele, whichallele is provided in a corresponding column in Table 14 next to thepeptide sequence.

TABLE 14 Peptide Sequences Comprising RAS G13DMutation, Corresponding HLA Allele, and Rank of Binding PotentialRank of Binding Peptide Allele Potential AGDVGKSAL HLA-C08:02 1AGDVGKSAL HLA-C05:01 2 VVGAGDVGK HLA-A11:01 3 VVVGAGDVGK HLA-All:01 3VVVGAGDVGK HLA-A68:01 4 GAGDVGKSA HLA-B46:01 5 GAGDVGKSAL HLA-B48:01 5VVGAGDVGK HLA-A68:01 5 VVGAGDVGK HLA-A03:01 5 AGDVGKSAL HLA-C03:04 6AGDVGKSAL HLA-C04:01 6 AGDVGKSAL HLA-C01:02 6 DVGKSALTI HLA-B13:02 6DVGKSALTI HLA-A25:01 6 GDVGKSAL HLA-C07:01 6 GDVGKSAL HLA-B40:02 7GDVGKSAL HLA-B37:01 8 AGDVGKSAL HLA-B48:01 9 DVGKSALTI HLA-B51:01 10VVGAGDVGK HLA-A30:01 10 GAGDVGKSAL HLA-C08:01 11 GAGDVGKSAL HLA-B81:0111 AGDVGKSAL HLA-C08:01 12 GAGDVGKSAL HLA-C03:04 12 DVGKSALTI HLA-B53:0113 AGDVGKSAL HLA-B07:02 14 AGDVGKSAL HLA-B46:01 14 DVGKSALTI HLA-A26:0114 VVGAGDVGK HLA-A74:01 14 GAGDVGKSA HLA-B54:01 15 DVGKSALTI HLA-B38:0116 GAGDVGKSAL HLA-C03:03 16 VVVGAGDVGK HLA-A03:01 16

A. Peptide Modification

In some embodiments, the present disclosure includes modified peptides.A modification can include a covalent chemical modification that doesnot alter the primary amino acid sequence of the antigenic peptideitself. Modifications can produce peptides with desired properties, forexample, prolonging the in vivo half-life, increasing the stability,reducing the clearance, altering the immunogenicity or allergenicity,enabling the raising of particular antibodies, cellular targeting,antigen uptake, antigen processing, HLA affinity, HLA stability orantigen presentation. In some embodiments, a peptide may comprise one ormore sequences that enhance processing and presentation of epitopes byAPCs, for example, for generation of an immune response.

In some embodiments, the peptide may be modified to provide desiredattributes. For instance, the ability of the peptides to induce CTLactivity can be enhanced by linkage to a sequence which contains atleast one epitope that is capable of inducing a T helper cell response.In some embodiments, immunogenic peptides/T helper conjugates are linkedby a spacer molecule. In some embodiments, a spacer comprises relativelysmall, neutral molecules, such as amino acids or amino acid mimetics,which are substantially uncharged under physiological conditions.Spacers can be selected from, e.g., Ala, Gly, or other neutral spacersof nonpolar amino acids or neutral polar amino acids. It will beunderstood that the optionally present spacer need not be comprised ofthe same residues and thus may be a hetero- or homo-oligomer. Theneoantigenic peptide may be linked to the T helper peptide eitherdirectly or via a spacer either at the amino or carboxy terminus of thepeptide. The amino terminus of either the neoantigenic peptide or the Thelper peptide may be acylated. Examples of T helper peptides includetetanus toxoid residues 830-843, influenza residues 307-319, and malariacircumsporozoite residues 382-398 and residues 378-389.

The peptide sequences of the present disclosure may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the peptide at preselected bases such that codons aregenerated that will translate into the desired amino acids.

In some embodiments, the peptide described herein can containsubstitutions to modify a physical property (e.g., stability orsolubility) of the resulting peptide. For example, the peptides can bemodified by the substitution of a cysteine (C) with α-amino butyric acid(“B”). Due to its chemical nature, cysteine has the propensity to formdisulfide bridges and sufficiently alter the peptide structurally so asto reduce binding capacity. Substituting α-amino butyric acid for C notonly alleviates this problem, but actually improves binding andcross-binding capability in certain instances. Substitution of cysteinewith α-amino butyric acid can occur at any residue of a neoantigenicpeptide, e.g., at either anchor or non-anchor positions of an epitope oranalog within a peptide, or at other positions of a peptide.

The peptide may also be modified by extending or decreasing thecompound's amino acid sequence, e.g., by the addition or deletion ofamino acids. The peptides or analogs can also be modified by alteringthe order or composition of certain residues. It will be appreciated bythe skilled artisan that certain amino acid residues essential forbiological activity, e.g., those at critical contact sites or conservedresidues, may generally not be altered without an adverse effect onbiological activity. The non-critical amino acids need not be limited tothose naturally occurring in proteins, such as L-α-amino acids, or theirD-isomers, but may include non-natural amino acids as well, such asβ-γ-δ-amino acids, as well as many derivatives of L-α-amino acids.

In some embodiments, the peptide may be modified using a series ofpeptides with single amino acid substitutions to determine the effect ofelectrostatic charge, hydrophobicity, etc. on HLA binding. For instance,a series of positively charged (e.g., Lys or Arg) or negatively charged(e.g., Glu) amino acid substitutions may be made along the length of thepeptide revealing different patterns of sensitivity towards various HLAmolecules and T cell receptors. In addition, multiple substitutionsusing small, relatively neutral moieties such as Ala, Gly, Pro, orsimilar residues may be employed. The substitutions may behomo-oligomers or hetero-oligomers. The number and types of residueswhich are substituted or added depend on the spacing necessary betweenessential contact points and certain functional attributes which aresought (e.g., hydrophobicity versus hydrophilicity). Increased bindingaffinity for an HLA molecule or T cell receptor may also be achieved bysuch substitutions, compared to the affinity of the parent peptide. Inany event, such substitutions should employ amino acid residues or othermolecular fragments chosen to avoid, for example, steric and chargeinterference which might disrupt binding. Amino acid substitutions aretypically of single residues. Substitutions, deletions, insertions orany combination thereof may be combined to arrive at a final peptide.

In some embodiments, the peptide described herein can comprise aminoacid mimetics or unnatural amino acid residues, e.g. D- orL-naphylalanine; D- or L-phenylglycine; D- or L-2-thieneylalanine; D- orL-1, -2, 3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoro-methyl)-phenylalanine;D-ρ-fluorophenylalanine; D- or L-ρ-biphenyl-phenylalanine; D- orL-ρ-methoxybiphenylphenylalanine; D- or L-2-indole(allyl) alanines; and,D- or L-alkylalanines, where the alkyl group can be a substituted orunsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl,iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino acid residues.Aromatic rings of a non-natural amino acid include, e.g., thiazolyl,thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, andpyridyl aromatic rings. Modified peptides that have various amino acidmimetics or unnatural amino acid residues may have increased stabilityin vivo. Such peptides may also have improved shelf-life ormanufacturing properties.

In some embodiments, a peptide described herein can be modified byterminal-NH₂ acylation, e.g., by alkanoyl (C₁-C₂₀) or thioglycolylacetylation, terminal-carboxyl amidation, e.g., ammonia, methylamine,etc. In some embodiments these modifications can provide sites forlinking to a support or other molecule. In some embodiments, the peptidedescribed herein can contain modifications such as but not limited toglycosylation, side chain oxidation, biotinylation, phosphorylation,addition of a surface active material, e.g. a lipid, or can bechemically modified, e.g., acetylation, etc. Moreover, bonds in thepeptide can be other than peptide bonds, e.g., covalent bonds, ester orether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.

In some embodiments, a peptide described herein can comprise carrierssuch as those well known in the art, e.g., thyroglobulin, albumins suchas human serum albumin, tetanus toxoid, polyamino acid residues such aspoly L-lysine and poly L-glutamic acid, influenza virus proteins,hepatitis B virus core protein, and the like.

The peptides can be further modified to contain additional chemicalmoieties not normally part of a protein. Those derivatized moieties canimprove the solubility, the biological half-life, absorption of theprotein, or binding affinity. The moieties can also reduce or eliminateany desirable side effects of the peptides and the like. An overview forthose moieties can be found in Remington's Pharmaceutical Sciences, 20thed., Mack Publishing Co., Easton, Pa. (2000). For example, neoantigenicpeptides having the desired activity may be modified as necessary toprovide certain desired attributes, e.g. improved pharmacologicalcharacteristics, while increasing or at least retaining substantiallyall of the biological activity of the unmodified peptide to bind thedesired HLA molecule and activate the appropriate T cell. For instance,the peptide may be subject to various changes, such as substitutions,either conservative or non-conservative, where such changes mightprovide for certain advantages in their use, such as improved HLAbinding. Such conservative substitutions may encompass replacing anamino acid residue with another amino acid residue that is biologicallyand/or chemically similar, e.g., one hydrophobic residue for another, orone polar residue for another. The effect of single amino acidsubstitutions may also be probed using D-amino acids. Such modificationsmay be made using well known peptide synthesis procedures, as describedin e.g., Merrifield, Science 232:341-347 (1986), Barany & Merrifield,The Peptides, Gross & Meienhofer, eds. (N.Y., Academic Press), pp. 1-284(1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford,III., Pierce), 2d Ed. (1984).

In some embodiments, the peptide described herein may be conjugated tolarge, slowly metabolized macromolecules such as proteins;polysaccharides, such as sepharose, agarose, cellulose, cellulose beads;polymeric amino acids such as polyglutamic acid, polylysine; amino acidcopolymers; inactivated virus particles; inactivated bacterial toxinssuch as toxoid from diphtheria, tetanus, cholera, leukotoxin molecules;inactivated bacteria; and dendritic cells.

Changes to the peptide that may include, but are not limited to,conjugation to a carrier protein, conjugation to a ligand, conjugationto an antibody, PEGylation, polysialylation HESylation, recombinant PEGmimetics, Fc fusion, albumin fusion, nanoparticle attachment,nanoparticulate encapsulation, cholesterol fusion, iron fusion,acylation, amidation, glycosylation, side chain oxidation,phosphorylation, biotinylation, the addition of a surface activematerial, the addition of amino acid mimetics, or the addition ofunnatural amino acids.

Glycosylation can affect the physical properties of proteins and canalso be important in protein stability, secretion, and subcellularlocalization. Proper glycosylation can be important for biologicalactivity. In fact, some genes from eukaryotic organisms, when expressedin bacteria (e.g., E. coli) which lack cellular processes forglycosylating proteins, yield proteins that are recovered with little orno activity by virtue of their lack of glycosylation. Addition ofglycosylation sites can be accomplished by altering the amino acidsequence. The alteration to the peptide or protein may be made, forexample, by the addition of, or substitution by, one or more serine orthreonine residues (for O-linked glycosylation sites) or asparagineresidues (for N-linked glycosylation sites). The structures of N-linkedand O-linked oligosaccharides and the sugar residues found in each typemay be different. One type of sugar that is commonly found on both isN-acetylneuraminic acid (hereafter referred to as sialic acid). Sialicacid is usually the terminal residue of both N-linked and O-linkedoligosaccharides and, by virtue of its negative charge, may conferacidic properties to the glycoprotein. Embodiments of the presentdisclosure comprise the generation and use of N-glycosylation variants.Removal of carbohydrates may be accomplished chemically orenzymatically, or by substitution of codons encoding amino acid residuesthat are glycosylated. Chemical deglycosylation techniques are known,and enzymatic cleavage of carbohydrate moieties on polypeptides can beachieved by the use of a variety of endo- and exo-glycosidases.

Additional suitable components and molecules for conjugation include,for example, molecules for targeting to the lymphatic system,thyroglobulin; albumins such as human serum albumin (HAS); tetanustoxoid; Diphtheria toxoid; polyamino acids such aspoly(D-lysine:D-glutamic acid); VP6 polypeptides of rotaviruses;influenza virus hemagglutinin, influenza virus nucleoprotein; KeyholeLimpet Hemocyanin (KLH); and hepatitis B virus core protein and surfaceantigen; or any combination of the foregoing.

Another type of modification is to conjugate (e.g., link) one or moreadditional components or molecules at the N- and/or C-terminus of apolypeptide sequence, such as another protein (e.g., a protein having anamino acid sequence heterologous to the subject protein), or a carriermolecule. Thus, an exemplary polypeptide sequence can be provided as aconjugate with another component or molecule. In some embodiments,fusion of albumin to the peptide or protein of the present disclosurecan, for example, be achieved by genetic manipulation, such that the DNAcoding for HSA, or a fragment thereof, is joined to the DNA coding forthe one or more polypeptide sequences. Thereafter, a suitable host canbe transformed or transfected with the fused nucleotide sequences in theform of, for example, a suitable plasmid, so as to express a fusionpolypeptide. The expression may be effected in vitro from, for example,prokaryotic or eukaryotic cells, or in vivo from, for example, atransgenic organism. In some embodiments of the present disclosure, theexpression of the fusion protein is performed in mammalian cell lines,for example, CHO cell lines. Furthermore, albumin itself may be modifiedto extend its circulating half-life. Fusion of the modified albumin toone or more polypeptides can be attained by the genetic manipulationtechniques described above or by chemical conjugation; the resultingfusion molecule has a half-life that exceeds that of fusions withnon-modified albumin (see, e.g., WO2011/051489). Several albumin-bindingstrategies have been developed as alternatives for direct fusion,including albumin binding through a conjugated fatty acid chain(acylation). Because serum albumin is a transport protein for fattyacids, these natural ligands with albumin-binding activity have beenused for half-life extension of small protein therapeutics.

Additional candidate components and molecules for conjugation includethose suitable for isolation or purification. Non-limiting examplesinclude binding molecules, such as biotin (biotin-avidin specificbinding pair), an antibody, a receptor, a ligand, a lectin, or moleculesthat comprise a solid support, including, for example, plastic orpolystyrene beads, plates or beads, magnetic beads, test strips, andmembranes. Purification methods such as cation exchange chromatographymay be used to separate conjugates by charge difference, whicheffectively separates conjugates into their various molecular weights.The content of the fractions obtained by cation exchange chromatographymay be identified by molecular weight using conventional methods, forexample, mass spectroscopy, SDS-PAGE, or other known methods forseparating molecular entities by molecular weight.

In some embodiments, the amino- or carboxyl-terminus of the peptide orprotein sequence of the present disclosure can be fused with animmunoglobulin Fc region (e.g., human Fc) to form a fusion conjugate (orfusion molecule). Fc fusion conjugates have been shown to increase thesystemic half-life of biopharmaceuticals, and thus the biopharmaceuticalproduct may require less frequent administration. Fc binds to theneonatal Fc receptor (FcRn) in endothelial cells that line the bloodvessels, and, upon binding, the Fc fusion molecule is protected fromdegradation and re-released into the circulation, keeping the moleculein circulation longer. This Fc binding is believed to be the mechanismby which endogenous IgG retains its long plasma half-life. More recentFc-fusion technology links a single copy of a biopharmaceutical to theFc region of an antibody to optimize the pharmacokinetic andpharmacodynamics properties of the biopharmaceutical as compared totraditional Fc-fusion conjugates.

The present disclosure contemplates the use of other modifications,currently known or developed in the future, of the peptides to improveone or more properties. One such method for prolonging the circulationhalf-life, increasing the stability, reducing the clearance, or alteringthe immunogenicity or allergenicity of the peptide of the presentdisclosure involves modification of the peptide sequences by hesylation,which utilizes hydroxyethyl starch derivatives linked to other moleculesin order to modify the molecule's characteristics. Various aspects ofhesylation are described in, for example, U.S. Patent Appln. Nos.2007/0134197 and 2006/0258607.

Peptide stability can be assayed in a number of ways. For instance,peptidases and various biological media, such as human plasma and serum,have been used to test stability. See, e.g., Verhoef, et al., Eur. J.Drug Me tab. Pharmacokinetics 11:291 (1986). Half-life of the peptidesdescribed herein is conveniently determined using a 25% human serum(v/v) assay. The protocol is as follows: pooled human serum (Type AB,non-heat inactivated) is dilapidated by centrifugation before use. Theserum is then diluted to 25% with RPMI-1640 or another suitable tissueculture medium. At predetermined time intervals, a small amount ofreaction solution is removed and added to either 6% aqueoustrichloroacetic acid (TCA) or ethanol. The cloudy reaction sample iscooled (4° C.) for 15 minutes and then spun to pellet the precipitatedserum proteins. The presence of the peptides is then determined byreversed-phase HPLC using stability-specific chromatography conditions.

Issues associated with short plasma half-life or susceptibility toprotease degradation may be overcome by various modifications, includingconjugating or linking the peptide or protein sequence to any of avariety of non-proteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes (see, for example, typicallyvia a linking moiety covalently bound to both the protein and thenonproteinaceous polymer, e.g., a PEG). Such PEG conjugated biomoleculeshave been shown to possess clinically useful properties, includingbetter physical and thermal stability, protection against susceptibilityto enzymatic degradation, increased solubility, longer in vivocirculating half-life and decreased clearance, reduced immunogenicityand antigenicity, and reduced toxicity.

PEGs suitable for conjugation to a polypeptide or protein sequence aregenerally soluble in water at room temperature, and have the generalformula R—(O—CH₂—CH₂)_(n)—O—R, where R is hydrogen or a protective groupsuch as an alkyl or an alkanol group, and where n is an integer from 1to 1000. When R is a protective group, it generally has from 1 to 8carbons. The PEG conjugated to the polypeptide sequence can be linear orbranched. Branched PEG derivatives, “star-PEGs” and multi-armed PEGs arecontemplated by the present disclosure. The present disclosure alsocontemplates compositions of conjugates wherein the PEGs have differentn values and thus the various different PEGs are present in specificratios. For example, some compositions comprise a mixture of conjugateswhere n=1, 2, 3 and 4. In some compositions, the percentage ofconjugates where n=1 is 18-25%, the percentage of conjugates where n=2is 50-66%, the percentage of conjugates where n=3 is 12-16%, and thepercentage of conjugates where n=4 is up to 5%. Such compositions can beproduced by reaction conditions and purification methods know in theart. For example, cation exchange chromatography may be used to separateconjugates, and a fraction is then identified which contains theconjugate having, for example, the desired number of PEGs attached,purified free from unmodified protein sequences and from conjugateshaving other numbers of PEGs attached.

PEG may be bound to the peptide or protein of the present disclosure viaa terminal reactive group (a “spacer”). The spacer is, for example, aterminal reactive group which mediates a bond between the free amino orcarboxyl groups of one or more of the polypeptide sequences and PEG. ThePEG having the spacer which may be bound to the free amino groupincludes N-hydroxysuccinylimide PEG which may be prepared by activatingsuccinic acid ester of PEG with N-hydroxysuccinylimide. Anotheractivated PEG which may be bound to a free amino group is2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine which may beprepared by reacting PEG monomethyl ether with cyanuric chloride. Theactivated PEG which is bound to the free carboxyl group includespolyoxyethylenediamine.

Conjugation of one or more of the peptide or protein sequences of thepresent disclosure to PEG having a spacer may be carried out by variousconventional methods. For example, the conjugation reaction can becarried out in solution at a pH of from 5 to 10, at temperature from 4°C. to room temperature, for 30 minutes to 20 hours, utilizing a molarratio of reagent to peptide/protein of from 4:1 to 30:1. Reactionconditions may be selected to direct the reaction towards producingpredominantly a desired degree of substitution. In general, lowtemperature, low pH (e.g., pH=5), and short reaction time tend todecrease the number of PEGs attached, whereas high temperature, neutralto high pH (e.g., pH>7), and longer reaction time tend to increase thenumber of PEGs attached. Various means known in the art may be used toterminate the reaction. In some embodiments the reaction is terminatedby acidifying the reaction mixture and freezing at, e.g., −20° C.

The present disclosure also contemplates the use of PEG mimetics.Recombinant PEG mimetics have been developed that retain the attributesof PEG (e.g., enhanced serum half-life) while conferring severaladditional advantageous properties. By way of example, simplepolypeptide chains (comprising, for example, Ala, Glu, Gly, Pro, Ser andThr) capable of forming an extended conformation similar to PEG can beproduced recombinantly already fused to the peptide or protein drug ofinterest (e.g., Amunix XTEN technology; Mountain View, Calif.). Thisobviates the need for an additional conjugation step during themanufacturing process. Moreover, established molecular biologytechniques enable control of the side chain composition of thepolypeptide chains, allowing optimization of immunogenicity andmanufacturing properties.

B. Neoepitopes

A neoepitope comprises a neoantigenic determinant part of a neoantigenicpeptide or neoantigenic polypeptide that is recognized by immune system.A neoepitope refers to an epitope that is not present in a reference,such as a non-diseased cell, e.g., a non-cancerous cell or a germlinecell, but is found in a diseased cell, e.g., a cancer cell. Thisincludes situations where a corresponding epitope is found in a normalnon-diseased cell or a germline cell but, due to one or more mutationsin a diseased cell, e.g., a cancer cell, the sequence of the epitope ischanged so as to result in the neoepitope. The term “neoepitope” is usedinterchangeably with “tumor specific neoepitope” in the presentspecification to designate a series of residues, typically L-aminoacids, connected one to the other, typically by peptide bonds betweenthe α-amino and carboxyl groups of adjacent amino acids. The neoepitopecan be a variety of lengths, either in their neutral (uncharged) formsor in forms which are salts, and either free of modifications such asglycosylation, side chain oxidation, or phosphorylation or containingthese modifications, subject to the condition that the modification notdestroy the biological activity of the polypeptides as herein described.The present disclosure provides isolated neoepitopes that comprise atumor specific mutation from Tables 1 to 14.

In some embodiments, neoepitopes described herein for HLA Class I are 13residues or less in length and usually consist of between about 8 andabout 12 residues, particularly 9 or 10 residues. In some embodiments,neoepitopes described herein for HLA Class II are 25 residues or less inlength and usually consist of between about 16 and about 25 residues.

In some embodiments, the composition described herein comprises a firstpeptide comprising a first neoepitope of a protein and a second peptidecomprising a second neoepitope of the same protein, wherein the firstpeptide is different from the second peptide, and wherein the firstneoepitope comprises a mutation and the second neoepitope comprises thesame mutation. In some embodiments, the composition described hereincomprises a first peptide comprising a first neoepitope of a firstregion of a protein and a second peptide comprising a second neoepitopeof a second region of the same protein, wherein the first regioncomprises at least one amino acid of the second region, wherein thefirst peptide is different from the second peptide and wherein the firstneoepitope comprises a first mutation and the second neoepitopecomprises a second mutation. In some embodiments, the first mutation andthe second mutation are the same. In some embodiments, the mutation isselected from the group consisting of a point mutation, a splice-sitemutation, a frameshift mutation, a read-through mutation, a gene fusionmutation and any combination thereof.

In some embodiments, the first neoepitope binds to a class I HLA proteinto form a class I HLA-peptide complex. In some embodiments, the secondneoepitope binds to a class II HLA a protein to form a class IIHLA-peptide complex. In some embodiments, the second neoepitope binds toa class I HLA protein to form a class I HLA-peptide complex. In someembodiments, the first neoepitope binds to a class II HLA protein toform a class II HLA-peptide complex. In some embodiments, the firstneoepitope activates CD8⁺ T cells. In some embodiments, the firstneoepitope activates CD4⁺ T cells. In some embodiments, the secondneoepitope activates CD4⁺ T cells. In some embodiments, the secondneoepitope activates CD8⁺ T cells. In some embodiments, a TCR of a CD4⁺T cell binds to a class II HLA-peptide complex. In some embodiments, aTCR of a CD8⁺ T cell binds to a class II HLA-peptide complex. In someembodiments, a TCR of a CD8⁺ T cell binds to a class I HLA-peptidecomplex. In some embodiments, a TCR of a CD4⁺ T cell binds to a class IHLA-peptide complex.

In some embodiments, the second neoepitope is longer than the firstneoepitope. In some embodiments, the first neoepitope has a length of atleast 8 amino acids. In some embodiments, the first neoepitope has alength of from 8 to 12 amino acids. In some embodiments, the firstneoepitope comprises a sequence of at least 8 contiguous amino acids,wherein at least 1 of the 8 contiguous amino acids are different atcorresponding positions of a wild-type sequence. In some embodiments,the first neoepitope comprises a sequence of at least 8 contiguous aminoacids, wherein at least 2 of the 8 contiguous amino acids are differentat corresponding positions of a wild-type sequence. In some embodiments,the second neoepitope has a length of at least 16 amino acids. In someembodiments, the second neoepitope has a length of from 16 to 25 aminoacids. In some embodiments, the second neoepitope comprises a sequenceof at least 16 contiguous amino acids, wherein at least 1 of the 16contiguous amino acids are different at corresponding positions of awild-type sequence. In some embodiments, the second neoepitope comprisesa sequence of at least 16 contiguous amino acids, wherein at least 2 ofthe 16 contiguous amino acids are different at corresponding positionsof a wild-type sequence.

In some embodiments, the neoepitope comprises at least one anchorresidue. In some embodiments, the first neoepitope, the secondneoepitope or both comprises at least one anchor residue. In oneembodiment, the at least one anchor residue of the first neoepitope isat a canonical anchor position or a non-canonical anchor position. Inanother embodiment, the at least one anchor residue of the secondneoepitope is at a canonical anchor position or a non-canonical anchorposition. In yet another embodiment, the at least one anchor residue ofthe first neoepitope is different from the at least one anchor residueof the second neoepitope.

In some embodiments, the at least one anchor residue is a wild-typeresidue. In some embodiments, the at least one anchor residue is asubstitution. In some embodiments, at least one anchor residue does notcomprise the mutation.

In some embodiments, the second neoepitope or both comprise at least oneanchor residue flanking region. In some embodiments, the neoepitopecomprises at least one anchor residue. In some embodiments, the at leastone anchor residues comprises at least two anchor residues. In someembodiments, the at least two anchor residues are separated by aseparation region comprising at least 1 amino acid. In some embodiments,the at least one anchor residue flanking region is not within theseparation region. In some embodiments, the at least one anchor residueflanking region is (a) upstream of a N-terminal anchor residue of the atleast two anchor residues; (b) downstream of a C-terminal anchor residueof the at least two anchor residues; or both (a) and (b).

In some embodiments, the neoepitopes bind an HLA protein (e.g., HLAclass I or HLA class II). In some embodiments, the neoepitopes bind anHLA protein with greater affinity than the corresponding wild-typepeptide. In some embodiments, the neoepitope has an IC₅₀ of less than5,000 nM, less than 1,000 nM, less than 500 nM, less than 100 nM, lessthan 50 nM, or less.

In some embodiments, the neoepitope can have an HLA binding affinity ofbetween about 1 pM and about 1 mM, about 100 pM and about 500 μM, about500 pM and about 10 μM, about 1 nM and about 1 μM, or about 10 nM andabout 1 μM. In some embodiments, the neoepitope can have an HLA bindingaffinity of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 700, 800, 900, or 1,000 nM, or more. Insome embodiments, the neoepitope can have an HLA binding affinity of atmost 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 700, 800, 900, or 1,000 nM.

In some embodiments, the first and/or second neoepitope binds to an HLAprotein with a greater affinity than a corresponding wild-typeneoepitope. In some embodiments, the first and/or second neoepitopebinds to an HLA protein with a K_(D) or an IC₅₀ less than 1,000 nM, 900nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nMor 10 nM. In some embodiments, the first and/or second neoepitope bindsto an HLA class I protein with a K_(D) or an IC₅₀ less than 1,000 nM,900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM,25 nM or 10 nM. In some embodiments, the first and/or second neoepitopebinds to an HLA class II protein with a K_(D) or an IC₅₀ less than 2,000nM, 1,500 nM, 1,000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM,150 nM, 100 nM, 50 nM, 25 nM or 10 nM.

In an aspect, the first and/or second neoepitope binds to a proteinencoded by an HLA allele expressed by a subject. In another aspect, themutation is not present in non-cancer cells of a subject. In yet anotheraspect, the first and/or second neoepitope is encoded by a gene or anexpressed gene of a subject's cancer cells.

In some embodiments, the first neoepitope comprises a mutation asdepicted in column 2 of Table 1 or 2 or column 1 of Tables 3 to 14. Insome embodiments, the second neoepitope comprises a mutation as depictedin column 2 of Table 1 or 2 or column 1 of Tables 3 to 14. In someembodiments, the first neoepitope and the second neoepitope is derivedfrom a TMPRSS2:ERG fusion protein. In some embodiments, the firstneoepitope and the second neoepitope is derived from a TMPRSS2:ERGfusion protein comprising a sequence of S::E from the sequenceMALNS::EALSVVSEDQSLFECAYGTPHLAKTEMTASSSSDYGQTSKMSPRVPQQDWALNSEALS V. Forexample, the first neoepitope and the second neoepitope can comprise asequence ALNSEALSVV. For example, the first neoepitope and the secondneoepitope can comprise a sequence MALNSEALSV.

In some embodiments, the first neoepitope and the second neoepitope isderived from a KRAS protein. In some embodiments, the first neoepitopeand the second neoepitope is derived from a NRAS protein. In someembodiments, the first neoepitope and the second neoepitope is derivedfrom a KRAS protein comprising a mutation of G12C, G12D, G12V, Q61H orQ61L substitution. In some embodiments, the first neoepitope and thesecond neoepitope is derived from a NRAS protein comprising a mutationof Q61K or Q61R substitution. In some embodiments, the neoepitopecomprises a substitution mutation, e.g., the KRAS G12C, G12D, G12V, Q61Hor Q61L mutation, or the NRAS Q61K or Q61R mutation. In someembodiments, the first neoepitope and the second neoepitope is derivedfrom a KRAS or NRAS protein sequence ofMTEYKLVVVGACGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQE. Forexample, the first neoepitope and the second neoepitope can comprise asequence KLVVVGACGV. For example, the first neoepitope and the secondneoepitope can comprise a sequence LVVVGACGV. For example, the firstneoepitope and the second neoepitope can comprise a sequence VVGACGVGK.For example, the first neoepitope and the second neoepitope can comprisea sequence VVVGACGVGK. In some embodiments, the first neoepitope and thesecond neoepitope is derived from a KRAS or NRAS protein sequence ofMTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEVVGADGVGK. For example, the first neoepitope and the second neoepitope cancomprise a sequence VVVGADGVGK. For example, the first neoepitope andthe second neoepitope can comprise a sequence KLVVVGADGV. For example,the first neoepitope and the second neoepitope can comprise a sequenceLVVVGADGV.

In some embodiments, the first neoepitope and the second neoepitope isderived from a KRAS or NRAS protein sequence ofMTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQE. Forexample, the first neoepitope and the second neoepitope can comprise asequence KLVVVGAVGV. For example, the first neoepitope and the secondneoepitope can comprise a sequence LVVVGAVGV. For example, the firstneoepitope and the second neoepitope can comprise a sequence VVGAVGVGK.For example, the first neoepitope and the second neoepitope can comprisea sequence VVVGAVGVGK.

In some embodiments, the first neoepitope and the second neoepitope isderived from a KRAS or NRAS protein sequence ofAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGHEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPM. For example, the first neoepitopeand the second neoepitope can comprise a sequence ILDTAGHEEY.

In some embodiments, the first neoepitope and the second neoepitope isderived from a KRAS or NRAS protein sequence ofAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGLEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPM. For example, the first neoepitopeand the second neoepitope can comprise a sequence ILDTAGLEEY. Forexample, the first neoepitope and the second neoepitope can comprise asequence LLDILDTAGL.

In some embodiments, the first neoepitope and the second neoepitope isderived from a KRAS or NRAS protein sequence ofAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGKEEYSAMRDQYMRTGEGFLCVFAINNSKSFADINLYREQIKRVKDSDDVPM. For example, the first neoepitopeand the second neoepitope can comprise a sequence ILDTAGKEEY.

In some embodiments, the first neoepitope and the second neoepitope isderived from a KRAS or NRAS protein sequence ofAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGREEYSAMRDQYMRTGEGFLCVFAINNSKSFADINLYREQIKRVKDSDDVPM. For example, the first neoepitopeand the second neoepitope can comprise a sequence ILDTAGREEY.

In some embodiments, the neoepitope comprises a sequence selected from agroup consisting of: DTAGHEEY, TAGHEEYSAM, DILDTAGHE, DILDTAGH,ILDTAGHEE, ILDTAGHE, DILDTAGHEEY, DTAGHEEYS, LLDILDTAGH, DILDTAGRE,DILDTAGR, ILDTAGREE, ILDTAGRE, CLLDILDTAGR, TAGREEYSAM, REEYSAMRD,DTAGKEEYSAM, CLLDILDTAGK, DTAGKEEY, LLDILDTAGK, ILDTAGKE, ILDTAGKEE,DTAGLEEY, ILDTAGLE, DILDTAGL, ILDTAGLEE, GLEEYSAMRDQY, LLDILDTAGLE,LDILDTAGL, DILDTAGLE, DILDTAGLEEY, AGVGKSAL, GAAGVGKSAL, AAGVGKSAL,CGVGKSAL, ACGVGKSAL, DGVGKSAL, ADGVGKSAL, DGVGKSALTI, GARGVGKSA,KLVVVGARGV, VVVGARGV, SGVGKSAL, VVVGASGVGK, GASGVGKSAL, VGVGKSAL,VVVGAGCVGK, KLVVVGAGC, GDVGKSAL, DVGKSALTI, VVVGAGDVGK, TAGKEEYSAM,DTAGHEEYSAM, TAGHEEYSA, DTAGREEYSAM, TAGKEEYSA, AAGVGKSA, AGCVGKSAL,AGDVGKSAL, AGKEEYSAMR, AGVGKSALTI, ARGVGKSAL, ASGVGKSA, ASGVGKSAL,AVGVGKSA, CVGKSALTI, DILDTAGK, DILDTAGREEY, DTAGHEEYSAMR, DTAGKEEYS,DTAGKEEYSAMR, DTAGLEEYS, DTAGLEEYSA, DTAGLEEYSAMR, DTAGREEYS,DTAGREEYSAMR, GAAGVGKSA, GACGVGKSA, GACGVGKSAL, GADGVGKS, GAGDVGKSA,GAGDVGKSAL, GASGVGKSA, GCVGKSAL, GCVGKSALTI, GHEEYSAM, GKEEYSAM,GLEEYSAMR, GREEYSAM, GREEYSAMR, HEEYSAMRD, KEEYSAMRD, KLVVVGASG,LDILDTAGR, LEEYSAMRD, LVVVGARGV, LVVVGASGV, REEYSAMRDQY, RGVGKSAL,TAGLEEYSA, TEYKLVVVGAA, VGAAGVGKSA, VGADGVGK, VGASGVGKSA, VGVGKSALTI,VVVGAAGV, VVVGAVGV, YKLVVVGAC, YKLVVVGAD, YKLVVVGAR, and DILDTAGKE.

The substitution may be positioned anywhere along the length of theneoepitope. For example, it can be located in the N terminal third ofthe peptide, the central third of the peptide or the C terminal third ofthe peptide. In another embodiment, the substituted residue is located2-5 residues away from the N terminal end or 2-5 residues away from theC terminal end. The peptides can be similarly derived from tumorspecific insertion mutations where the peptide comprises one or more, orall of the inserted residues.

In some embodiments, the peptide as described herein can be readilysynthesized chemically utilizing reagents that are free of contaminatingbacterial or animal substances (Merrifield R B: Solid phase peptidesynthesis. I. The synthesis of a tetrapeptide. J. Am. Chem.Soc.85:2149-54, 1963). In some embodiments, peptides are prepared by (1)parallel solid-phase synthesis on multi-channel instruments usinguniform synthesis and cleavage conditions; (2) purification over aRP-HPLC column with column stripping; and re-washing, but notreplacement, between peptides; followed by (3) analysis with a limitedset of the most informative assays. The Good Manufacturing Practices(GMP) footprint can be defined around the set of peptides for anindividual patient, thus requiring suite changeover procedures onlybetween syntheses of peptides for different patients.

C. Polynucleotides

Alternatively, a nucleic acid (e.g., a polynucleotide) encoding thepeptide of the present disclosure may be used to produce theneoantigenic peptide in vitro. The polynucleotide may be, e.g., DNA,cDNA, PNA, CNA, RNA, either single- and/or double-stranded, or native orstabilized forms of polynucleotides, such as e.g. polynucleotides with aphosphorothiate backbone, or combinations thereof and it may or may notcontain introns so long as it codes for the peptide. In some embodimentsin vitro translation is used to produce the peptide.

Provided herein are neoantigenic polynucleotides encoding each of theneoantigenic peptides described in the present disclosure. The term“polynucleotide”, “nucleotides” or “nucleic acid” is usedinterchangeably with “mutant polynucleotide”, “mutant nucleotide”,“mutant nucleic acid”, “neoantigenic polynucleotide”, “neoantigenicnucleotide” or “neoantigenic mutant nucleic acid” in the presentdisclosure. Various nucleic acid sequences can encode the same peptidedue to the redundancy of the genetic code. Each of these nucleic acidsfalls within the scope of the present disclosure. Nucleic acids encodingpeptides can be DNA or RNA, for example, mRNA, or a combination of DNAand RNA. In some embodiments, a nucleic acid encoding a peptide is aself-amplifying mRNA (Brito et al., Adv. Genet. 2015; 89:179-233). Anysuitable polynucleotide that encodes a peptide described herein fallswithin the scope of the present disclosure.

The term “RNA” includes and in some embodiments relates to “mRNA.” Theterm “mRNA” means “messenger-RNA” and relates to a “transcript” which isgenerated by using a DNA template and encodes a peptide or polypeptide.Typically, an mRNA comprises a 5′-UTR, a protein coding region, and a3′-UTR. mRNA only possesses limited half-life in cells and in vitro. Insome embodiments, the mRNA is self-amplifying mRNA. In the context ofthe present disclosure, mRNA may be generated by in vitro transcriptionfrom a DNA template. The in vitro transcription methodology is known tothe skilled person. For example, there is a variety of in vitrotranscription kits commercially available.

The stability and translation efficiency of RNA may be modified asrequired. For example, RNA may be stabilized and its translationincreased by one or more modifications having a stabilizing effectsand/or increasing translation efficiency of RNA. Such modifications aredescribed, for example, in PCT/EP2006/009448, incorporated herein byreference. In order to increase expression of the RNA used according tothe present disclosure, it may be modified within the coding region,i.e., the sequence encoding the expressed peptide or protein, withoutaltering the sequence of the expressed peptide or protein, so as toincrease the GC-content to increase mRNA stability and to perform acodon optimization and, thus, enhance translation in cells.

The term “modification” in the context of the RNA used in the presentdisclosure includes any modification of an RNA which is not naturallypresent in said RNA. In some embodiments, the RNA does not have uncapped5′-triphosphates. Removal of such uncapped 5′-triphosphates can beachieved by treating RNA with a phosphatase. In other embodiments, theRNA may have modified ribonucleotides in order to increase its stabilityand/or decrease cytotoxicity. In some embodiments, 5-methylcytidine canbe substituted partially or completely in the RNA, for example, forcytidine. Alternatively, pseudouridine is substituted partially orcompletely, for example, for uridine.

In some embodiments, the term “modification” relates to providing an RNAwith a 5′-cap or 5′-cap analog. The term “5′-cap” refers to a capstructure found on the 5′-end of an mRNA molecule and generally consistsof a guanosine nucleotide connected to the mRNA via an unusual 5′ to 5′triphosphate linkage. In some embodiments, this guanosine is methylatedat the 7-position. The term “conventional 5′-cap” refers to a naturallyoccurring RNA 5′-cap, to the 7-methylguanosine cap (m G). In the contextof the present disclosure, the term “5′-cap” includes a 5′-cap analogthat resembles the RNA cap structure and is modified to possess theability to stabilize RNA and/or enhance translation of RNA if attachedthereto, in vivo and/or in a cell.

In certain embodiments, an mRNA encoding a neoantigenic peptide of thepresent disclosure is administered to a subject in need thereof. In someembodiments, the present disclosure provides RNA, oligoribonucleotide,and polyribonucleotide molecules comprising a modified nucleoside, genetherapy vectors comprising same, gene therapy methods and genetranscription silencing methods comprising same. In some embodiments,the mRNA to be administered comprises at least one modified nucleoside.

The polynucleotides encoding peptides described herein can besynthesized by chemical techniques, for example, the phosphotriestermethod of Matteucci, et al., J. Am. Chem. Soc. 103:3185 (1981).Polynucleotides encoding peptides comprising or consisting of an analogcan be made simply by substituting the appropriate and desired nucleicacid base(s) for those that encode the native epitope.

Polynucleotides described herein can comprise one or more synthetic ornaturally-occurring introns in the transcribed region. The inclusion ofmRNA stabilization sequences and sequences for replication in mammaliancells can also be considered for increasing polynucleotide expression.In addition, a polynucleotide described herein can compriseimmunostimulatory sequences (ISSs or CpGs). These sequences can beincluded in the vector, outside the polynucleotide coding sequence toenhance immunogenicity.

In some embodiments, the polynucleotides may comprise the codingsequence for the peptide or protein fused in the same reading frame to apolynucleotide which aids, for example, in expression and/or secretionof the peptide or protein from a host cell (e.g., a leader sequencewhich functions as a secretory sequence for controlling transport of apolypeptide from the cell). The polypeptide having a leader sequence isa pre-protein and can have the leader sequence cleaved by the host cellto form the mature form of the polypeptide.

In some embodiments, the polynucleotides can comprise the codingsequence for the peptide or protein fused in the same reading frame to amarker sequence that allows, for example, for purification of theencoded peptide, which may then be incorporated into a personalizeddisease vaccine or immunogenic composition. For example, the markersequence can be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or the marker sequence can be ahemagglutinin (HA) tag derived from the influenza hemagglutinin proteinwhen a mammalian host (e.g., COS-7 cells) is used. Additional tagsinclude, but are not limited to, Calmodulin tags, FLAG tags, Myc tags, Stags, SBP tags, Softag 1, Softag 3, V5 tag, Xpress tag, Isopeptag,SpyTag, Biotin Carboxyl Carrier Protein (BCCP) tags, GST tags,fluorescent protein tags (e.g., green fluorescent protein tags), maltosebinding protein tags, Nus tags, Strep-tag, thioredoxin tag, TC tag, Tytag, and the like.

In some embodiments, the polynucleotides may comprise the codingsequence for one or more the presently described peptides or proteinsfused in the same reading frame to create a single concatamerizedneoantigenic peptide construct capable of producing multipleneoantigenic peptides.

In some embodiments, a DNA sequence is constructed using recombinanttechnology by isolating or synthesizing a DNA sequence encoding awild-type protein of interest. Optionally, the sequence can bemutagenized by site-specific mutagenesis to provide functional analogsthereof. See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA81:5662-5066 (1984) and U.S. Pat. No. 4,588,585. In another embodiment,a DNA sequence encoding the peptide or protein of interest would beconstructed by chemical synthesis using an oligonucleotide synthesizer.Such oligonucleotides can be designed based on the amino acid sequenceof the desired peptide and selecting those codons that are favored inthe host cell in which the recombinant polypeptide of interest isproduced. Standard methods can be applied to synthesize an isolatedpolynucleotide sequence encoding an isolated polypeptide of interest.For example, a complete amino acid sequence can be used to construct aback-translated gene. Further, a DNA oligomer containing a nucleotidesequence coding for the particular isolated polypeptide can besynthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly

Once assembled (e.g., by synthesis, site-directed mutagenesis, oranother method), the polynucleotide sequences encoding a particularisolated polypeptide of interest is inserted into an expression vectorand optionally operatively linked to an expression control sequenceappropriate for expression of the protein in a desired host. Properassembly can be confirmed by nucleotide sequencing, restriction mapping,and expression of a biologically active polypeptide in a suitable host.As well known in the art, in order to obtain high expression levels of atransfected gene in a host, the gene can be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

Thus, the present disclosure is also directed to vectors, and expressionvectors useful for the production and administration of the neoantigenicpeptides and neoepitopes described herein, and to host cells comprisingsuch vectors.

IV. Vectors

In some embodiments, an expression vector capable of expressing thepeptide or protein as described herein can also be prepared. Expressionvectors for different cell types are well known in the art and can beselected without undue experimentation. Generally, the DNA is insertedinto an expression vector, such as a plasmid, in proper orientation andcorrect reading frame for expression. If necessary, the DNA may belinked to the appropriate transcriptional and translational regulatorycontrol nucleotide sequences recognized by the desired host (e.g.,bacteria), although such controls are generally available in theexpression vector. The vector is then introduced into the host bacteriafor cloning using standard techniques (see, e.g., Sambrook et al. (1989)Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.).

A large number of vectors and host systems suitable for producing andadministering a neoantigenic peptide described herein are known to thoseof skill in the art, and are commercially available. The followingvectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9(Qiagen), pBS, pD10, phagescript, psiX174, pBluescript SK, pbsks, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia); pCR (Invitrogen). Eukaryotic: pWLNEO,pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL(Pharmacia); p75.6 (Valentis); pCEP (Invitrogen); pCEI (Epimmune).However, any other plasmid or vector can be used as long as it isreplicable and viable in the host.

For expression of the neoantigenic peptides described herein, the codingsequence will be provided operably linked start and stop codons,promoter and terminator regions, and in some embodiments, and areplication system to provide an expression vector for expression in thedesired cellular host. For example, promoter sequences compatible withbacterial hosts are provided in plasmids containing convenientrestriction sites for insertion of the desired coding sequence. Theresulting expression vectors are transformed into suitable bacterialhosts.

Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences. Such promoters can also be derived from viral sources, suchas, e.g., human cytomegalovirus (CMV-IE promoter) or herpes simplexvirus type-1 (HSV TK promoter). Nucleic acid sequences derived from theSV40 splice, and polyadenylation sites can be used to provide therequired nontranscribed genetic elements.

Recombinant expression vectors may be used to amplify and express DNAencoding the peptide or protein as described herein. Recombinantexpression vectors are replicable DNA constructs which have synthetic orcDNA-derived DNA fragments encoding a peptide or a bioequivalent analogoperatively linked to suitable transcriptional or translationalregulatory elements derived from mammalian, microbial, viral or insectgenes. A transcriptional unit generally comprises an assembly of (1) agenetic element or elements having a regulatory role in gene expression,for example, transcriptional promoters or enhancers, (2) a structural orcoding sequence which is transcribed into mRNA and translated intoprotein, and (3) appropriate transcription and translation initiationand termination sequences, as described in detail herein. Suchregulatory elements can include an operator sequence to controltranscription. The ability to replicate in a host, usually conferred byan origin of replication, and a selection gene to facilitate recognitionof transformants can additionally be incorporated. DNA regions areoperatively linked when they are functionally related to each other. Forexample, DNA for a signal peptide (secretory leader) is operativelylinked to DNA for a polypeptide if it is expressed as a precursor whichparticipates in the secretion of the polypeptide; a promoter isoperatively linked to a coding sequence if it controls the transcriptionof the sequence; or a ribosome binding site is operatively linked to acoding sequence if it is positioned so as to permit translation.Generally, operatively linked means contiguous, and in the case ofsecretory leaders, means contiguous and in reading frame. Structuralelements intended for use in yeast expression systems include a leadersequence enabling extracellular secretion of translated protein by ahost cell. Alternatively, where recombinant protein is expressed withouta leader or transport sequence, it can include an N-terminal methionineresidue. This residue can optionally be subsequently cleaved from theexpressed recombinant protein to provide a final product.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), acid phosphatase, or heat shockproteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and in some embodiments, a leader sequencecapable of directing secretion of translated protein into theperiplasmic space or extracellular medium. Optionally, the heterologoussequence can encode a fusion protein including an N-terminalidentification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct.

Polynucleotides encoding neoantigenic peptides described herein can alsocomprise a ubiquitination signal sequence, and/or a targeting sequencesuch as an endoplasmic reticulum (ER) signal sequence to facilitatemovement of the resulting peptide into the endoplasmic reticulum.

In some embodiments, the neoantigenic peptide described herein can alsobe administered and/or expressed by viral or bacterial vectors. Examplesof expression vectors include attenuated viral hosts, such as vacciniaor fowlpox. As an example of this approach, vaccinia virus is used as avector to express nucleotide sequences that encode the neoantigenicpeptides described herein. Vaccinia vectors and methods useful inimmunization protocols are described in, e.g., U.S. Pat. No. 4,722,848.Another vector is BCG (Bacille Calmette Guerin). BCG vectors aredescribed by Stover et al., Nature 351:456-460 (1991).

A wide variety of other vectors useful for therapeutic administration orimmunization of the neoantigenic polypeptides described herein, e.g.adeno and adeno-associated virus vectors, retroviral vectors, SalmonellaTyphimurium vectors, detoxified anthrax toxin vectors, Sendai virusvectors, poxvirus vectors, canarypox vectors, and fowlpox vectors, andthe like, will be apparent to those skilled in the art from thedescription herein. In some embodiments, the vector is Modified VacciniaAnkara (VA) (e.g. Bavarian Noridic (MVA-BN)).

Among vectors that may be used in the practice of the presentdisclosure, integration in the host genome of a cell is possible withretrovirus gene transfer methods, often resulting in long termexpression of the inserted transgene. In some embodiments, theretrovirus is a lentivirus. Additionally, high transduction efficiencieshave been observed in many different cell types and target tissues. Thetropism of a retrovirus can be altered by incorporating foreign envelopeproteins, expanding the potential target population of target cells. Aretrovirus can also be engineered to allow for conditional expression ofthe inserted transgene, such that only certain cell types are infectedby the lentivirus. Cell type specific promoters can be used to targetexpression in specific cell types. Lentiviral vectors are retroviralvectors (and hence both lentiviral and retroviral vectors may be used inthe practice of the present disclosure). Moreover, lentiviral vectorsare able to transduce or infect non-dividing cells and typically producehigh viral titers. Selection of a retroviral gene transfer system maytherefore depend on the target tissue. Retroviral vectors are comprisedof cis-acting long terminal repeats with packaging capacity for up to6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficientfor replication and packaging of the vectors, which are then used tointegrate the desired nucleic acid into the target cell to providepermanent expression. Widely used retroviral vectors that may be used inthe practice of the present disclosure include those based upon murineleukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., (1992) J. Virol.66:2731-2739; Johann et al., (1992) J. Virol. 66:1635-1640; Sommnerfeltet al., (1990) Virol. 176:58-59; Wilson et al., (1998) J. Virol.63:2374-2378; Miller et al., (1991) J. Virol. 65:2220-2224;PCT/US94/05700).

Also useful in the practice of the present disclosure is a minimalnon-primate lentiviral vector, such as a lentiviral vector based on theequine infectious anemia virus (EIAV). The vectors may havecytomegalovirus (CMV) promoter driving expression of the target gene.Accordingly, the present disclosure contemplates amongst vector(s)useful in the practice of the present disclosure: viral vectors,including retroviral vectors and lentiviral vectors.

Also useful in the practice of the present disclosure is an adenovirusvector. One advantage is the ability of recombinant adenoviruses toefficiently transfer and express recombinant genes in a variety ofmammalian cells and tissues in vitro and in vivo, resulting in the highexpression of the transferred nucleic acids. Further, the ability toproductively infect quiescent cells, expands the utility of recombinantadenoviral vectors. In addition, high expression levels ensure that theproducts of the nucleic acids will be expressed to sufficient levels togenerate an immune response (see e.g., U.S. Pat. No. 7,029,848, herebyincorporated by reference).

As to adenovirus vectors useful in the practice of the presentdisclosure, mention is made of U.S. Pat. No. 6,955,808. The adenovirusvector used can be selected from the group consisting of the Ad5, Ad35,Ad11, C6, and C7 vectors. The sequence of the Adenovirus 5 (“Ad5”)genome has been published. (Chroboczek, J., Bieber, F., and Jacrot, B.(1992) The Sequence of the Genome of Adenovirus Type 5 and ItsComparison with the Genome of Adenovirus Type 2, Virology 186, 280-285;the contents if which is hereby incorporated by reference). Ad35 vectorsare described in U.S. Pat. Nos. 6,974,695, 6,913,922, and 6,869,794.Ad11 vectors are described in U.S. Pat. No. 6,913,922. C6 adenovirusvectors are described in U.S. Pat. Nos. 6,780,407; 6,537,594; 6,309,647;6,265,189; 6,156,567; 6,090,393; 5,942,235 and 5,833,975. C7 vectors aredescribed in U.S. Pat. No. 6,277,558. Adenovirus vectors that areE1-defective or deleted, E3-defective or deleted, and/or E4-defective ordeleted may also be used. Certain adenoviruses having mutations in theE1 region have improved safety margin because E1-defective adenovirusmutants are replication-defective in non-permissive cells, or, at thevery least, are highly attenuated. Adenoviruses having mutations in theE3 region may have enhanced the immunogenicity by disrupting themechanism whereby adenovirus down-regulates MHC class I molecules.Adenoviruses having E4 mutations may have reduced immunogenicity of theadenovirus vector because of suppression of late gene expression. Suchvectors may be particularly useful when repeated re-vaccinationutilizing the same vector is desired. Adenovirus vectors that aredeleted or mutated in E1, E3, E4; E1 and E3; and E1 and E4 can be usedin accordance with the present disclosure.

Furthermore, “gutless” adenovirus vectors, in which all viral genes aredeleted, can also be used in accordance with the present disclosure.Such vectors require a helper virus for their replication and require aspecial human 293 cell line expressing both Ela and Cre, a conditionthat does not exist in natural environment. Such “gutless” vectors arenon-immunogenic and thus the vectors may be inoculated multiple timesfor re-vaccination. The “gutless” adenovirus vectors can be used forinsertion of heterologous inserts/genes such as the transgenes of thepresent disclosure, and can even be used for co-delivery of a largenumber of heterologous inserts/genes.

In some embodiments, the delivery is via an adenovirus, which may be ata single booster dose. In some embodiments, the adenovirus is deliveredvia multiple doses. In terms of in vivo delivery, AAV is advantageousover other viral vectors due to low toxicity and low probability ofcausing insertional mutagenesis because it doesn't integrate into thehost genome. AAV has a packaging limit of 4.5 or 4.75 Kb. Constructslarger than 4.5 or 4.75 Kb result in significantly reduced virusproduction. There are many promoters that can be used to drive nucleicacid molecule expression. AAV ITR can serve as a promoter and isadvantageous for eliminating the need for an additional promoterelement.

For ubiquitous expression, the following promoters can be used: CMV,CAG, CBh, PGK, SV40, Ferritin heavy or light chains, etc. For brainexpression, the following promoters can be used: Synapsin I for allneurons, CaMK II alpha for excitatory neurons, GAD67 or GAD65 or VGATfor GABAergic neurons, etc. Promoters used to drive RNA synthesis caninclude: Pol III promoters such as U6 or H1. The use of a Pol IIpromoter and intronic cassettes can be used to express guide RNA (gRNA).With regard to AAV vectors useful in the practice of the presentdisclosure, mention is made of U.S. Pat. Nos. 5,658,785, 7,115,391,7,172,893, 6,953,690, 6,936,466, 6,924,128, 6,893,865, 6,793,926,6,537,540, 6,475,769 and 6,258,595, and documents cited therein. As toAAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof. One canselect the AAV with regard to the cells to be targeted; e.g., one canselect AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or anycombination thereof for targeting brain or neuronal cells; and one canselect AAV4 for targeting cardiac tissue. AAV8 is useful for delivery tothe liver. In some embodiments the delivery is via an AAV. The dosagemay be adjusted to balance the therapeutic benefit against any sideeffects.

In some embodiments, a Poxvirus is used in the presently describedcomposition. These include orthopoxvirus, avipox, vaccinia, MVA, NYVAC,canarypox, ALVAC, fowlpox, TROVAC, etc. (see e.g., Verardiet al., Hum.Vaccin. Immunother. 2012 July; 8(7):961-70; and Moss, Vaccine. 2013;31(39): 4220-4222). Poxvirus expression vectors were described in 1982and quickly became widely used for vaccine development as well asresearch in numerous fields. Advantages of the vectors include simpleconstruction, ability to accommodate large amounts of foreign DNA andhigh expression levels. Information concerning poxviruses that may beused in the practice of the present disclosure, such as Chordopoxvirinaesubfamily poxviruses (poxviruses of vertebrates), for instance,orthopoxviruses and avipoxviruses, e.g., vaccinia virus (e.g., WyethStrain, WR Strain (e.g., ATCC® VR-1354), Copenhagen Strain, NYVAC,NYVAC.1, NYVAC.2, MVA, MVA-BN), canarypox virus (e.g., Wheatley C93Strain, ALVAC), fowlpox virus (e.g., FP9 Strain, Webster Strain,TROVAC), dovepox, pigeonpox, quailpox, and raccoon pox, inter alia,synthetic or non-naturally occurring recombinants thereof, uses thereof,and methods for making and using such recombinants may be found inscientific and patent literature.

In some embodiments, the vaccinia virus is used in the disease vaccineor immunogenic composition to express a antigen. (Rolph et al.,Recombinant viruses as vaccines and immunological tools. Curr. Opin.Immunol. 9:517-524, 1997). The recombinant vaccinia virus is able toreplicate within the cytoplasm of the infected host cell and thepolypeptide of interest can therefore induce an immune response.Moreover, Poxviruses have been widely used as vaccine or immunogeniccomposition vectors because of their ability to target encoded antigensfor processing by the major histocompatibility complex class I pathwayby directly infecting immune cells, in particular antigen-presentingcells, but also due to their ability to self-adjuvant.

In some embodiments, ALVAC is used as a vector in a disease vaccine orimmunogenic composition. ALVAC is a canarypox virus that can be modifiedto express foreign transgenes and has been used as a method forvaccination against both prokaryotic and eukaryotic antigens (Horig H,Lee D S, Conkright W, et al. Phase I clinical trial of a recombinantcanarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigenand the B7.1 co-stimulatory molecule. Cancer Immunol. Immunother. 2000;49:504-14; von Mehren M, Arlen P, Tsang K Y, et al. Pilot study of adual gene recombinant avipox vaccine containing both carcinoembryonicantigen (CEA) and B7.1 transgenes in patients with recurrentCEA-expressing adenocarcinomas. Clin. Cancer. Res. 2000; 6:2219-28;Musey L, Ding Y, Elizaga M, et al. HIV-1 vaccination administeredintramuscularly can induce both systemic and mucosal T cell immunity inHIV-1-uninfected individuals. J. Immunol. 2003; 171:1094-101; PaolettiE. Applications of pox virus vectors to vaccination: an update. Proc.Natl. Acad. Sci. USA 1996; 93:11349-53; U.S. Pat. No. 7,255,862). In aphase I clinical trial, an ALVAC virus expressing the tumor antigen CEAshowed an excellent safety profile and resulted in increasedCEA-specific T cell responses in selected patients; objective clinicalresponses, however, were not observed (Marshall J L, Hawkins M J, TsangK Y, et al. Phase I study in cancer patients of a replication-defectiveavipox recombinant vaccine that expresses human carcinoembryonicantigen. J. Clin. Oncol. 1999; 17:332-7).

In some embodiments, a Modified Vaccinia Ankara (MVA) virus may be usedas a viral vector for an antigen vaccine or immunogenic composition. MVAis a member of the Orthopoxvirus family and has been generated by about570 serial passages on chicken embryo fibroblasts of the Ankara strainof Vaccinia virus (CVA) (see, e.g., Mayr, A., et al., Infection 3, 6-14,1975). As a consequence of these passages, the resulting MVA viruscontains 31 kilobases less genomic information compared to CVA, and ishighly host cell restricted (Meyer, H. et al., J. Gen. Virol. 72,1031-1038, 1991). MVA is characterized by its extreme attenuation,namely, by a diminished virulence or infectious ability, but still holdsan excellent immunogenicity. When tested in a variety of animal models,MVA was proven to be avirulent, even in immuno-suppressed individuals.Moreover, MVA-BN®-HER2 is a candidate immunotherapy designed for thetreatment of HER-2-positive breast cancer and is currently in clinicaltrials. (Mandl et al., Cancer Immunol. Immunother. January 2012; 61(1):19-29). Methods to make and use recombinant MVA has been described(e.g., see U.S. Pat. Nos. 8,309,098 and 5,185,146 hereby incorporated inits entirety).

Suitable host cells for expression of a polypeptide include prokaryotes,yeast, insect or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or bacilli. Higher eukaryoticcells include established cell lines of mammalian origin. Cell-freetranslation systems could also be employed. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are well known in the art (see Pouwels et al., CloningVectors: A Laboratory Manual, Elsevier, N.Y., 1985).

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175,1981), and other cell lines capable of expressing an appropriate vectorincluding, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO),293, HeLa and BHK cell lines. Mammalian expression vectors can comprisenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 3′ flanking nontranscribed sequences, and 5′ or 3′ nontranslatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences. Baculovirus systems for production of heterologous proteinsin insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47(1988).

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors which can be, for example, a cloningvector or an expression vector. The vector can be, for example, in theform of a plasmid, a viral particle, a phage, etc. The engineered hostcells can be cultured in conventional nutrient media modified asappropriate for activating promoters, selecting transformants oramplifying the polynucleotides. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

As representative examples of appropriate hosts, there can be mentioned:bacterial cells, such as E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus; fungal cells, such as yeast; insectcells such as Drosophila and Sf9; animal cells such as COS-7 lines ofmonkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), andother cell lines capable of expressing a compatible vector, for example,the C127, 3T3, CHO, HeLa and BHK cell lines or Bowes melanoma; plantcells, etc. The selection of an appropriate host is deemed to be withinthe scope of those skilled in the art from the teachings herein.

Yeast, insect or mammalian cell hosts can also be used, employingsuitable vectors and control sequences. Examples of mammalian expressionsystems include the COS-7 lines of monkey kidney fibroblasts, describedby Gluzman, Cell 23:175 (1981), and other cell lines capable ofexpressing a compatible vector, for example, the C127, 3T3, CHO, HeLaand BHK cell lines.

Polynucleotides described herein can be administered and expressed inhuman cells (e.g., immune cells, including dendritic cells). A humancodon usage table can be used to guide the codon choice for each aminoacid. Such polynucleotides comprise spacer amino acid residues betweenepitopes and/or analogs, such as those described above, or can comprisenaturally-occurring flanking sequences adjacent to the epitopes and/oranalogs (and/or CTL (e.g., CD8⁺), Th (e.g., CD4⁺), and B cell epitopes).

Standard regulatory sequences well known to those of skill in the artcan be included in the vector to ensure expression in the human targetcells. Several vector elements are desirable: a promoter with adownstream cloning site for polynucleotide, e.g., minigene insertion; apolyadenylation signal for efficient transcription termination; an E.coli origin of replication; and an E. coli selectable marker (e.g.ampicillin or kanamycin resistance). Numerous promoters can be used forthis purpose, e.g., the human cytomegalovirus (hCMV) promoter. See,e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promotersequences. In some embodiments, the promoter is the CMV-IE promoter.

Useful expression vectors for eukaryotic hosts, especially mammals orhumans include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Escherichia coli,including pCR1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Vectors may be introduced into animal tissues by a number of differentmethods. The two most popular approaches are injection of DNA in saline,using a standard hypodermic needle, and gene gun delivery. A schematicoutline of the construction of a DNA vaccine plasmid and its subsequentdelivery by these two methods into a host is illustrated at ScientificAmerican (Weiner et al., (1999) Scientific American 281 (1): 34-41).Injection in saline is normally conducted intramuscularly (IM) inskeletal muscle, or intradermally (ID), with DNA being delivered to theextracellular spaces. This can be assisted by electroporation bytemporarily damaging muscle fibers with myotoxins such as bupivacaine;or by using hypertonic solutions of saline or sucrose (Alarcon et al.,(1999). Adv. Parasitol. Advances in Parasitology 42: 343-410). Immuneresponses to this method of delivery can be affected by many factors,including needle type, needle alignment, speed of injection, volume ofinjection, muscle type, and age, sex and physiological condition of theanimal being injected(Alarcon et al., (1999). Adv. Parasitol. Advancesin Parasitology 42: 343-410).

Gene gun delivery, the other commonly used method of delivery,ballistically accelerates plasmid DNA (pDNA) that has been adsorbed ontogold or tungsten microparticles into the target cells, using compressedhelium as an accelerant (Alarcon et al., (1999). Adv. Parasitol.Advances in Parasitology 42: 343-410; Lewis et al., (1999). Advances inVirus Research (Academic Press) 54: 129-88).

Alternative delivery methods may include aerosol instillation of nakedDNA on mucosal surfaces, such as the nasal and lung mucosa, (Lewis etal., (1999). Advances in Virus Research (Academic Press) 54: 129-88) andtopical administration of pDNA to the eye and vaginal mucosa (Lewis etal., (1999) Advances in Virus Research (Academic Press) 54: 129-88).Mucosal surface delivery has also been achieved using cationicliposome-DNA preparations, biodegradable microspheres, attenuatedShigella or Listeria vectors for oral administration to the intestinalmucosa, and recombinant adenovirus vectors. DNA or RNA may also bedelivered to cells following mild mechanical disruption of the cellmembrane, temporarily permeabilizing the cells. Such a mild mechanicaldisruption of the membrane can be accomplished by gently forcing cellsthrough a small aperture (Sharei et al., Ex Vivo Cytosolic Delivery ofFunctional Macromolecules to Immune Cells, PLOS ONE (2015)).

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle). In the casewhere a non-viral delivery system is utilized, an exemplary deliveryvehicle is a liposome. “Liposome” is a generic term encompassing avariety of single and multilamellar lipid vehicles formed by thegeneration of enclosed lipid bilayers or aggregates. Liposomes can becharacterized as having vesicular structures with a phospholipid bilayermembrane and an inner aqueous medium. Multilamellar liposomes havemultiple lipid layers separated by aqueous medium. They formspontaneously when phospholipids are suspended in an excess of aqueoussolution. The lipid components undergo self-rearrangement before theformation of closed structures and entrap water and dissolved solutesbetween the lipid bilayers (Ghosh et al., Glycobiology 5: 505-10(1991)). However, compositions that have different structures insolution than the normal vesicular structure are also encompassed. Forexample, the lipids may assume a micellar structure or merely exist asnonuniform aggregates of lipid molecules. Also contemplated arelipofectamine-nucleic acid complexes.

The use of lipid formulations is contemplated for the introduction ofthe nucleic acids into a host cell (in vitro, ex vivo or in vivo). Inanother aspect, the nucleic acid may be associated with a lipid. Thenucleic acid associated with a lipid may be encapsulated in the aqueousinterior of a liposome, interspersed within the lipid bilayer of aliposome, attached to a liposome via a linking molecule that isassociated with both the liposome and the oligonucleotide, entrapped ina liposome, complexed with a liposome, dispersed in a solutioncontaining a lipid, mixed with a lipid, combined with a lipid, containedas a suspension in a lipid, contained or complexed with a micelle, orotherwise associated with a lipid. Lipid, lipid/DNA or lipid/expressionvector associated compositions are not limited to any particularstructure in solution. For example, they may be present in a bilayerstructure, as micelles, or with a “collapsed” structure. They may alsosimply be interspersed in a solution, possibly forming aggregates thatare not uniform in size or shape. Lipids are fatty substances which maybe naturally occurring or synthetic lipids. For example, lipids includethe fatty droplets that naturally occur in the cytoplasm as well as theclass of compounds which contain long-chain aliphatic hydrocarbons andtheir derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol.

In some embodiments, a vector comprises a polynucleotide encoding afirst peptide comprising a first neoepitope and a second peptidecomprising a second neoepitope. In some embodiments, the first andsecond peptides are derived from the same protein. The at least twodistinct peptides may vary by length, amino acid sequence or both. Thepeptides are derived from any protein known to or have been found tocontain a tumor specific mutation. In some embodiments, a vectorcomprises a first peptide comprising a first neoepitope of a protein anda second peptide comprising a second neoepitope of the same protein,wherein the first peptide is different from the second peptide, andwherein the first neoepitope comprises a mutation and the secondneoepitope comprises the same mutation. In some embodiments, a vectorcomprises a first peptide comprising a first neoepitope of a firstregion of a protein and a second peptide comprising a second neoepitopeof a second region of the same protein, wherein the first regioncomprises at least one amino acid of the second region, wherein thefirst peptide is different from the second peptide and wherein the firstneoepitope comprises a first mutation and the second neoepitopecomprises a second mutation. In some embodiments, the first mutation andthe second mutation are the same. In some embodiments, the mutation isselected from the group consisting of a point mutation, a splice-sitemutation, a frameshift mutation, a read-through mutation, a gene fusionmutation and any combination thereof.

In some embodiments, a vector comprises a polynucleotide operably linkedto a promoter. In some embodiments, the vector is a self-amplifying RNAreplicon, plasmid, phage, transposon, cosmid, virus, or virion. In someembodiments, the vector is derived from a retrovirus, lentivirus,adenovirus, adeno-associated virus, herpes virus, pox virus, alphavirus, vaccinia virus, hepatitis B virus, human papillomavirus or apseudotype thereof. In some embodiments, the vector is a non-viralvector. In some embodiments, the non-viral vector is a nanoparticle, acationic lipid, a cationic polymer, a metallic nanopolymer, a nanorod, aliposome, a micelle, a microbubble, a cell-penetrating peptide, or aliposphere.

V. T Cell Receptors

In one aspect, the present disclosure provides cells expressing aneoantigen-recognizing receptor that activates an immunoresponsive cell(e.g., T cell receptor (TCR) or chimeric antigen receptor (CAR)), andmethods of using such cells for the treatment of a disease that requiresan enhanced immune response. Such cells include genetically modifiedimmunoresponsive cells (e.g., T cells, Natural Killer (NK) cells,cytotoxic T lymphocytes (CTL (e.g., CD8⁺)) cells, helper T lymphocyte(Th (e.g., CD4⁺)) cells) expressing an antigen-recognizing receptor(e.g., TCR or CAR) that binds one of the neoantigenic peptides describedherein, and methods of use therefore for the treatment of neoplasia andother pathologies where an increase in an antigen-specific immuneresponse is desired. T cell activation is mediated by a TCR or a CARtargeted to an antigen.

The present disclosure provides cells expressing a combination of anantigen-recognizing receptor that activates an immunoresponsive cell(e.g., TCR, CAR) and a chimeric co-stimulating receptor (CCR), andmethods of using such cells for the treatment of a disease that requiresan enhanced immune response. In some embodiments, tumor antigen-specificT cells, NK cells, CTL cells or other immunoresponsive cells are used asshuttles for the selective enrichment of one or more co-stimulatoryligands for the treatment or prevention of neoplasia. Such cells areadministered to a human subject in need thereof for the treatment orprevention of a particular cancer.

In some embodiments, the tumor antigen-specific human lymphocytes thatcan be used in the methods of the present disclosure include, withoutlimitation, peripheral donor lymphocytes genetically modified to expresschimeric antigen receptors (CARs) (Sadelain, M., et al. 2003 Nat RevCancer 3:35-45), peripheral donor lymphocytes genetically modified toexpress a full-length tumor antigen-recognizing T cell receptor complexcomprising the a and p heterodimer (Morgan, R. A., et al. 2006 Science314:126-129), lymphocyte cultures derived from tumor infiltratinglymphocytes (TILs) in tumor biopsies (Panelli, M. C., et al. 2000 JImmunol 164:495-504; Panelli, M. C., et al. 2000 J Immunol164:4382-4392), and selectively in vitro-expanded antigen-specificperipheral blood leukocytes employing artificial antigen-presentingcells (AAPCs) or pulsed dendritic cells (Dupont, J., et al. 2005 CancerRes 65:5417-5427; Papanicolaou, G. A., et al. 2003 Blood 102:2498-2505).The T cells may be autologous, allogeneic, or derived in vitro fromengineered progenitor or stem cells.

In some embodiments, the immunotherapeutic is an engineered receptor. Insome embodiments, the engineered receptor is a chimeric antigen receptor(CAR), a T cell receptor (TCR), or a B-cell receptor (BCR), an adoptiveT cell therapy (ACT), or a derivative thereof. In other aspects, theengineered receptor is a chimeric antigen receptor (CAR). In someaspects, the CAR is a first generation CAR. In other aspects, the CAR isa second generation CAR. In still other aspects, the CAR is a thirdgeneration CAR. In some aspects, the CAR comprises an extracellularportion, a transmembrane portion, and an intracellular portion. In someaspects, the intracellular portion comprises at least one T cellco-stimulatory domain. In some aspects, the T cell co-stimulatory domainis selected from the group consisting of CD27, CD28, TNFRS9 (4-1BB),TNFRSF4 (OX40), TNFRSF8 (CD30), CD40LG (CD40L), ICOS, ITGB2 (LFA-1),CD2, CD7, KLRC2 (NKG2C), TNFRS18 (GITR), TNFRSF14 (HVEM), or anycombination thereof.

In some aspects, the engineered receptor binds a target. In someaspects, the binding is specific to a peptide specific to one or moresubjects suffering from a disease or condition.

In some aspects, the immunotherapeutic is a cell as described in detailherein. In some aspects, the immunotherapeutic is a cell comprising areceptor that specifically binds a peptide or neoepitope describedherein. In some aspects, the immunotherapeutic is a cell used incombination with the peptides/nucleic acids of the present disclosure.In some embodiments, the cell is a patient cell. In some embodiments,the cell is a T cell. In some embodiments, the cell is tumorinfiltrating lymphocyte.

In some aspects, a subject with a condition or disease is treated basedon a T cell receptor repertoire of the subject. In some embodiments, apeptide or neoepitope is selected based on a T cell receptor repertoireof the subject. In some embodiments, a subject is treated with T cellsexpressing TCRs specific to a peptide or neoepitope as described herein.In some embodiments, a subject is treated with a peptide or neoepitopespecific to TCRs, e.g., subject specific TCRs. In some embodiments, asubject is treated with a peptide or neoepitope specific to T cellsexpressing TCRs, e.g., subject specific TCRs. In some embodiments, asubject is treated with a peptide or neoepitope specific to subjectspecific TCRs.

In some embodiments, the composition as described herein is selectedbased on TCRs identified in one or more subjects. In some embodiments,identification of a T cell repertoire and testing in functional assaysis used to determine the composition to be administered to one or moresubjects with a condition or disease. In some embodiments, thecomposition is an antigen vaccine comprising one or more peptides orproteins as described herein. In some embodiments, the vaccine comprisessubject specific neoantigenic peptides. In some embodiments, thepeptides to be included in the vaccine are selected based on aquantification of subject specific TCRs that bind to the neoepitopes. Insome embodiments, the peptides are selected based on a binding affinityof the peptide to a TCR. In some embodiments, the selecting is based ona combination of both the quantity and the binding affinity. Forexample, a TCR that binds strongly to a neoepitope in a functionalassay, but that is not highly represented in a TCR repertoire may be agood candidate for an antigen vaccine because T cells expressing the TCRwould be advantageously amplified.

In some embodiments, the peptide or protein is selected foradministering to one or more subjects based on binding to TCRs. In someembodiments, T cells, such as T cells from a subject with a disease orcondition, can be expanded. Expanded T cells that express TCRs specificto a neoantigenic peptide or neoepitope can be administered back to asubject. In some embodiments, suitable cells, e.g., PBMCs, aretransduced or transfected with polynucleotides for expression of TCRsspecific to a neoantigenic peptide or neoepitope and administered to asubject. T cells expressing TCRs specific to a neoantigenic peptide orneoepitope can be expanded and administered back to a subject. In someembodiments, T cells that express TCRs specific to a neoantigenicpeptide or neoepitope that result in cytolytic activity when incubatedwith autologous diseased tissue can be expanded and administered to asubject. In some embodiments, T cells used in functional assays resultin binding to a neoantigenic peptide or neoepitope can be expanded andadministered to a subject. In some embodiments, TCRs that have beendetermined to bind to subject specific neoantigenic peptides orneoepitopes can be expressed in T cells and administered to a subject.

In an embodiment, the present disclosure provides a compositioncomprising a first peptide comprising a first neoepitope and a secondpeptide comprising a second neoepitope, wherein the first peptide isdifferent from the second peptide, and wherein the first neoepitopecomprises a mutation and the second neoepitope comprises the samemutation. In some embodiments, the composition as provided hereincomprises a first T cell comprising a first T cell receptor (TCR)specific for the first neoepitope and a second T cell comprising asecond TCR specific for the second neoepitope. In some embodiments, thefirst and second peptides are derived from the same protein.

In another embodiment, the present disclosure provides a compositioncomprising a first peptide comprising a first neoepitope of a firstregion of a protein and a second peptide comprising a second neoepitopeof a second region of the same protein, wherein the first regioncomprises at least one amino acid of the second region, wherein thefirst peptide is different from the second peptide and wherein the firstneoepitope comprises a first mutation and the second neoepitopecomprises a second mutation. In some embodiments, the composition asprovided herein comprises a first T cell comprising a first T cellreceptor (TCR) specific for the first neoepitope and a second T cellcomprising a second TCR specific for the second neoepitope. In someembodiments, the first mutation and the second mutation are the same.

In some embodiments, the first neoepitope binds to a class I HLA proteinto form a class I HLA-peptide complex. In some embodiments, the firstneoepitope binds to a class II HLA protein to form a class IIHLA-peptide complex. In some embodiments, the second neoepitope binds toa class II HLA a protein to form a class II HLA-peptide complex. In someembodiments, the second neoepitope binds to a class I HLA protein toform a class I HLA-peptide complex. In some embodiments, the firstneoepitope activates CD8⁺ T cells. In some embodiments, the firstneoepitope activates CD4⁺ T cells. In some embodiments, the secondneoepitope activates CD4⁺ T cells. In some embodiments, the secondneoepitope activates CD8⁺ T cells. In some embodiments, a TCR of a CD4⁺T cell binds to a class II HLA-peptide complex. In some embodiments, aTCR of a CD8⁺ T cell binds to a class II HLA-peptide complex. In someembodiments, a TCR of a CD8⁺ T cell binds to a class I HLA-peptidecomplex. In some embodiments, a TCR of a CD4⁺ T cell binds to a class IHLA-peptide complex.

In some embodiments, the first TCR is a first chimeric antigen receptorspecific for the first neoepitope and the second TCR is a secondchimeric antigen receptor specific for the second neoepitope. In someembodiments, the first T cell is a cytotoxic T cell. In someembodiments, the first T cell is a gamma delta T cell. In someembodiments, the second T cell is a helper T cell. In some embodiments,the first and/or second TCR binds to an HLA-peptide complex with a K_(D)or an IC₅₀ of less than 1,000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM or 10 nM. In some embodiments,the first and/or second TCR binds to an HLA class I-peptide complex witha K_(D) or an IC₅₀ of less than 1,000 nM, 900 nM, 800 nM, 700 nM, 600nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM or 10 nM. In someembodiments, the first and/or second TCR binds to an HLA classII-peptide complex with a K_(D) or an IC₅₀ of less than 2,000, 1,500,1,000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100nM, 50 nM, 25 nM or 10 nM.

VI. Antigen Presenting Cells

The neoantigenic peptide or protein can be provided as antigenpresenting cells (e.g., dendritic cells) containing such peptides,proteins or polynucleotides as described herein. In other embodiments,such antigen presenting cells are used to stimulate T cells for use inpatients. Thus, one embodiment of the present disclosure is acomposition containing at least one antigen presenting cell (e.g., adendritic cell) that is pulsed or loaded with one or more neoantigenicpeptides or polynucleotides described herein. In some embodiments, suchAPCs are autologous (e.g., autologous dendritic cells). Alternatively,peripheral blood mononuclear cells (PBMCs) isolated from a patient canbe loaded with neoantigenic peptides or polynucleotides ex vivo. Inrelated embodiments, such APCs or PBMCs are injected back into thepatient. In some embodiments, the antigen presenting cells are dendriticcells. In related embodiments, the dendritic cells are autologousdendritic cells that are pulsed with the neoantigenic peptide or nucleicacid. The neoantigenic peptide can be any suitable peptide that givesrise to an appropriate T cell response. T cell therapy using autologousdendritic cells pulsed with peptides from a tumor associated antigen isdisclosed in Murphy et al. (1996) The Prostate 29, 371-380 and Tjua etal. (1997) The Prostate 32, 272-278. In some embodiments, the T cell isa CTL (e.g., CD8⁺). In some embodiments, the T cell is a helper Tlymphocyte (Th (e.g., CD4⁺)).

In some embodiments, the present disclosure provides a compositioncomprising a cell-based immunogenic pharmaceutical composition that canalso be administered to a subject. For example, an antigen presentingcell (APC) based immunogenic pharmaceutical composition can beformulated using any of the well-known techniques, carriers, andexcipients as suitable and as understood in the art. APCs includemonocytes, monocyte-derived cells, macrophages, and dendritic cells.Sometimes, an APC based immunogenic pharmaceutical composition can be adendritic cell-based immunogenic pharmaceutical composition.

A dendritic cell-based immunogenic pharmaceutical composition can beprepared by any methods well known in the art. In some cases, dendriticcell-based immunogenic pharmaceutical compositions can be preparedthrough an ex vivo or in vivo method. The ex vivo method can comprisethe use of autologous DCs pulsed ex vivo with the polypeptides describedherein, to activate or load the DCs prior to administration into thepatient. The in vivo method can comprise targeting specific DC receptorsusing antibodies coupled with the polypeptides described herein. TheDC-based immunogenic pharmaceutical composition can further comprise DCactivators such as TLR3, TLR-7-8, and CD40 agonists. The DC-basedimmunogenic pharmaceutical composition can further comprise adjuvants,and a pharmaceutically acceptable carrier.

Antigen presenting cells (APCs) can be prepared from a variety ofsources, including human and non-human primates, other mammals, andvertebrates. In certain embodiments, APCs can be prepared from blood ofa human or non-human vertebrate. APCs can also be isolated from anenriched population of leukocytes. Populations of leukocytes can beprepared by methods known to those skilled in the art. Such methodstypically include collecting heparinized blood, apheresis orleukopheresis, preparation of buffy coats, rosetting, centrifugation,density gradient centrifugation (e.g., using Ficoll, colloidal silicaparticles, and sucrose), differential lysis non-leukocyte cells, andfiltration. A leukocyte population can also be prepared by collectingblood from a subject, defibrillating to remove the platelets and lysingthe red blood cells. The leukocyte population can optionally be enrichedfor monocytic dendritic cell precursors.

Blood cell populations can be obtained from a variety of subjects,according to the desired use of the enriched population of leukocytes.The subject can be a healthy subject. Alternatively, blood cells can beobtained from a subject in need of immunostimulation, such as, forexample, a cancer patient or other patient for which immunostimulationwill be beneficial. Likewise, blood cells can be obtained from a subjectin need of immune suppression, such as, for example, a patient having anautoimmune disorder (e.g., rheumatoid arthritis, diabetes, lupus,multiple sclerosis, and the like). A population of leukocytes also canbe obtained from an HLA-matched healthy individual.

When blood is used as a source of APC, blood leukocytes may be obtainedusing conventional methods that maintain their viability. According toone aspect of the present disclosure, blood can be diluted into mediumthat may or may not contain heparin or other suitable anticoagulant. Thevolume of blood to medium can be about 1 to 1. Cells can be concentratedby centrifugation of the blood in medium at about 1,000 rpm (150 g) at4° C. Platelets and red blood cells can be depleted by resuspending thecells in any number of solutions known in the art that will lyseerythrocytes, for example ammonium chloride. For example, the mixturemay be medium and ammonium chloride at about 1:1 by volume. Cells may beconcentrated by centrifugation and washed in the desired solution untila population of leukocytes, substantially free of platelets and redblood cells, is obtained. Any isotonic solution commonly used in tissueculture may be used as the medium for separating blood leukocytes fromplatelets and red blood cells. Examples of such isotonic solutions canbe phosphate buffered saline, Hanks balanced salt solution, and completegrowth media. APCs and/or APC precursor cells may also purified byelutriation.

In one embodiment, the APCs can be non-nominal APCs under inflammatoryor otherwise activated conditions. For example, non-nominal APCs caninclude epithelial cells stimulated with interferon-gamma, T cells, Bcells, and/or monocytes activated by factors or conditions that induceAPC activity. Such non-nominal APCs can be prepared according to methodsknown in the art.

The APCs can be cultured, expanded, differentiated and/or, matured, asdesired, according to the according to the type of APC. The APCs can becultured in any suitable culture vessel, such as, for example, cultureplates, flasks, culture bags, and bioreactors.

In certain embodiments, APCs can be cultured in suitable culture orgrowth medium to maintain and/or expand the number of APCs in thepreparation. The culture media can be selected according to the type ofAPC isolated. For example, mature APCs, such as mature dendritic cells,can be cultured in growth media suitable for their maintenance andexpansion. The culture medium can be supplemented with amino acids,vitamins, antibiotics, divalent cations, and the like. In addition,cytokines, growth factors and/or hormones, can be included in the growthmedia. For example, for the maintenance and/or expansion of maturedendritic cells, cytokines, such as granulocyte/macrophage colonystimulating factor (GM-CSF), FMS-like tyrosine kinase 3 ligand (FLT-3L)and/or interleukin 4 (IL-4), can be added. In other embodiments,immature APCs can be cultured and/or expanded. Immature dendritic cellscan they retain the ability to uptake target mRNA and process newantigen. In some embodiments, immature dendritic cells can be culturedin media suitable for their maintenance and culture. The culture mediumcan be supplemented with amino acids, vitamins, antibiotics, divalentcations, and the like. In addition, cytokines, growth factors and/orhormones, can be included in the growth media.

Other immature APCs can similarly be cultured or expanded. Preparationsof immature APCs can be matured to form mature APCs. Maturation of APCscan occur during or following exposure to the neoantigenic peptides. Incertain embodiments, preparations of immature dendritic cells can bematured. Suitable maturation factors include, for example, cytokinesTNF-α, bacterial products (e.g., BCG), and the like. In another aspect,isolated APC precursors can be used to prepare preparations of immatureAPCs. APC precursors can be cultured, differentiated, and/or matured. Incertain embodiments, monocytic dendritic cell precursors can be culturedin the presence of suitable culture media supplemented with amino acids,vitamins, cytokines, and/or divalent cations, to promote differentiationof the monocytic dendritic cell precursors to immature dendritic cells.In some embodiments, the APC precursors are isolated from PBMCs. ThePBMCs can be obtained from a donor, for example, a human donor, and canbe used freshly or frozen for future usage. In some embodiments, the APCis prepared from one or more APC preparations. In some embodiments, theAPC comprises an APC loaded with the first and second neoantigenicpeptides comprising the first and second neoepitopes or polynucleotidesencoding the first and second neoantigenic peptides comprising the firstand second neoepitopes. In some embodiments, the APC is an autologousAPC, an allogenic APC, or an artificial APC.

In an embodiment, the present disclosure provides a compositioncomprising an APC comprising a first peptide comprising a firstneoepitope and a second peptide comprising a second neoepitope, whereinthe first peptide is different from the second peptide, and wherein thefirst neoepitope comprises a mutation and the second neoepitopecomprises the same mutation. In some embodiments, the first and secondpeptides are derived from the same protein. In another embodiment, thepresent disclosure provides a composition comprising an APC comprising afirst peptide comprising a first neoepitope of a first region of aprotein and a second peptide comprising a second neoepitope of a secondregion of the same protein, wherein the first region comprises at leastone amino acid of the second region, wherein the first peptide isdifferent from the second peptide and wherein the first neoepitopecomprises a first mutation and the second neoepitope comprises a secondmutation. In some embodiments, the first mutation and the secondmutation are the same.

VII. Adjuvants

An adjuvant can be used to enhance the immune response (humoral and/orcellular) elicited in a patient receiving a composition as providedherein. Sometimes, adjuvants can elicit a Th1-type response. Othertimes, adjuvants can elicit a Th2-type response. A Th1-type response canbe characterized by the production of cytokines such as IFN-γ as opposedto a Th2-type response which can be characterized by the production ofcytokines such as IL-4, IL-5 and IL-10.

In some aspects, lipid-based adjuvants, such as MPLA and MDP, can beused with the immunogenic pharmaceutical compositions disclosed herein.Monophosphoryl lipid A (MPLA), for example, is an adjuvant that causesincreased presentation of liposomal antigen to specific T Lymphocytes.In addition, a muramyl dipeptide (MDP) can also be used as a suitableadjuvant in conjunction with the immunogenic pharmaceutical formulationsdescribed herein.

Suitable adjuvants are known in the art (see, WO 2015/095811) andinclude, but are not limited to poly(I:C), poly-ICLC, Hiltonol, STINGagonist, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893,CpG7909, CyaA, dSLIM, GM-CSF, FLT-3L, IC30, IC31, Imiquimod, ImuFactIMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®.vector system, PLG microparticles, resiquimod, SRL172, virosomes andother virus-like particles, YF-17D, VEGF trap, R848, beta-glucan,Pam3Cys, Pam3CSK4, Aquila's QS21 stimulon (Aquila Biotech, Worcester,Mass., USA) which is derived from saponin, mycobacterial extracts andsynthetic bacterial cell wall mimics, and other proprietary adjuvantssuch as Ribi's Detox. Quil or Superfos. Adjuvants also includeincomplete Freund's or GM-CSF. Several immunological adjuvants (e.g.,MF59) specific for dendritic cells and their preparation have beendescribed previously (Dupuis M, et al., Cell Immunol. 1998;186(1):18-27; Allison A C; Dev. Biol. Stand. 1998; 92:3-11) (Mosca etal. Frontiers in Bioscience, 2007; 12:4050-4060) (Gamvrellis et al.Immunol & Cell Biol. 2004; 82: 506-516). Also cytokines can be used.Several cytokines have been directly linked to influencing dendriticcell migration to lymphoid tissues (e.g., TNF-alpha), accelerating thematuration of dendritic cells into efficient antigen-presenting cellsfor T-lymphocytes (e.g., GM-CSF, FLT-3L, PGE1, PGE2, IL-1, IL-1b, IL-4,IL-6 and CD40L) (U.S. Pat. No. 5,849,589 incorporated herein byreference in its entirety) and acting as immunoadjuvants (e.g., IL-12)(Gabrilovich D I, et al., J. Immunother. Emphasis Tumor Immunol. 1996(6):414-418).

Adjuvant can also comprise stimulatory molecules such as cytokines.Non-limiting examples of cytokines include: CCL20, α-interferon(IFN-α),β-interferon (IFN-β), γ-interferon, platelet derived growth factor(PDGF), TNFα, TNFβ (lymphotoxin alpha (LTα)), GM-CSF, FLT-3L, epidermalgrowth factor (EGF), cutaneous T cell-attracting chemokine (CTACK),epithelial thymus-expressed chemokine (TECK), mucosae-associatedepithelial chemokine (MEC), IL-12, IL-15, IL-28, MHC, CD80, CD86, IL-1,IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-la, MIP-1-, IL-8,L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1,VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF,G-CSF, mutant forms of IL-18, CD40, CD40L, vascular growth factor,fibroblast growth factor, IL-7, nerve growth factor, vascularendothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1,DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2,DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88,IRAK, TRAF6, IκB, Inactive NIK, SAP K, SAP-I, JNK, interferon responsegenes, NFκB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4,RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B,NKG2C, NKG2E, NKG2F, TAPI, and TAP2.

Additional adjuvants include: MCP-1, MIP-la, MIP-1p, IL-8, RANTES,L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1,VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF,G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor,fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascularendothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1,DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2,DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88,IRAK, TRAF6, IκB, Inactive NIK, SAP K, SAP-1, JNK, interferon responsegenes, NFκB, Bax, TRAIL, 1RAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4,RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B,NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof.

In some aspects, an adjuvant can be a modulator of a toll like receptor.Examples of modulators of toll-like receptors include TLR-9 agonists andare not limited to small molecule modulators of toll-like receptors suchas Imiquimod. Other examples of adjuvants that are used in combinationwith an immunogenic pharmaceutical composition described herein caninclude and are not limited to saponin, CpG ODN and the like. Sometimes,an adjuvant is selected from bacteria toxoids,polyoxypropylene-polyoxyethylene block polymers, aluminum salts,liposomes, CpG polymers, oil-in-water emulsions, or a combinationthereof. Sometimes, an adjuvant is an oil-in-water emulsion. Theoil-in-water emulsion can include at least one oil and at least onesurfactant, with the oil(s) and surfactant(s) being biodegradable(metabolisable) and biocompatible. The oil droplets in the emulsion canbe less than 5 μm in diameter, and can even have a sub-micron diameter,with these small sizes being achieved with a microfluidiser to providestable emulsions. Droplets with a size less than 220 nm can be subjectedto filter sterilization.

VIII. Methods of Treatment and Pharmaceutical Compositions

The neoantigen therapeutics (e.g., peptides, polynucleotides, TCR, CAR,cells containing TCR or CAR, APC or dendritic cell containingpolypeptide, dendritic cell containing polynucleotide, antibody, etc.)described herein are useful in a variety of applications including, butnot limited to, therapeutic treatment methods, such as the treatment ofcancer. In some embodiments, the therapeutic treatment methods compriseimmunotherapy. In certain embodiments, a neoantigenic peptide is usefulfor activating, promoting, increasing, and/or enhancing an immuneresponse, redirecting an existing immune response to a new target,increasing the immunogenicity of a tumor, inhibiting tumor growth,reducing tumor volume, increasing tumor cell apoptosis, and/or reducingthe tumorigenicity of a tumor. The methods of use can be in vitro, exvivo, or in vivo methods.

In some aspects, the present disclosure provides methods for activatingan immune response in a subject using a neoantigenic peptide or proteindescribed herein. In some embodiments, the present disclosure providesmethods for promoting an immune response in a subject using aneoantigenic peptide described herein. In some embodiments, the presentdisclosure provides methods for increasing an immune response in asubject using a neoantigenic peptide described herein. In someembodiments, the present disclosure provides methods for enhancing animmune response using a neoantigenic peptide. In some embodiments, theactivating, promoting, increasing, and/or enhancing of an immuneresponse comprises increasing cell-mediated immunity. In someembodiments, the activating, promoting, increasing, and/or enhancing ofan immune response comprises increasing T cell activity or humoralimmunity. In some embodiments, the activating, promoting, increasing,and/or enhancing of an immune response comprises increasing CTL or Thactivity. In some embodiments, the activating, promoting, increasing,and/or enhancing of an immune response comprises increasing NK cellactivity. In some embodiments, the activating, promoting, increasing,and/or enhancing of an immune response comprises increasing T cellactivity and increasing NK cell activity. In some embodiments, theactivating, promoting, increasing, and/or enhancing of an immuneresponse comprises increasing CTL activity and increasing NK cellactivity. In some embodiments, the activating, promoting, increasing,and/or enhancing of an immune response comprises inhibiting ordecreasing the suppressive activity of T regulatory (Treg) cells. Insome embodiments, the immune response is a result of antigenicstimulation. In some embodiments, the antigenic stimulation is a tumorcell. In some embodiments, the antigenic stimulation is cancer.

In some embodiments, the present disclosure provides methods ofactivating, promoting, increasing, and/or enhancing of an immuneresponse using a neoantigenic peptide described herein. In someembodiments, a method comprises administering to a subject in needthereof a therapeutically effective amount of a neoantigenic peptidethat delivers a neoantigenic peptide or polynucleotide to a tumor cell.In some embodiments, a method comprises administering to a subject inneed thereof a therapeutically effective amount of a neoantigenicpeptide internalized by the tumor cell. In some embodiments, a methodcomprises administering to a subject in need thereof a therapeuticallyeffective amount of a neoantigenic peptide that is internalized by atumor cell, and the neoantigenic peptide is processed by the cell. Insome embodiments, a method comprises administering to a subject in needthereof a therapeutically effective amount of a neoantigenic polypeptidethat is internalized by a tumor cell and a neoepitope is presented onthe surface of the tumor cell. In some embodiments, a method comprisesadministering to a subject in need thereof a therapeutically effectiveamount of a neoantigenic polypeptide that is internalized by the tumorcell, is processed by the cell, and an antigenic peptide is presented onthe surface of the tumor cell.

In some embodiments, a method comprises administering to a subject inneed thereof a therapeutically effective amount of a neoantigenicpeptide or polynucleotide described herein that delivers an exogenouspolypeptide comprising at least one neoantigenic peptide to a tumorcell, wherein at least one neoepitope derived from the neoantigenicpeptide is presented on the surface of the tumor cell. In someembodiments, the antigenic peptide is presented on the surface of thetumor cell in complex with a MHC class I molecule. In some embodiments,the neoepitope is presented on the surface of the tumor cell in complexwith a MHC class II molecule.

In some embodiments, a method comprises contacting a tumor cell with aneoantigenic polypeptide or polynucleotide described herein thatdelivers an exogenous polypeptide comprising at least one neoantigenicpeptide to the tumor cell, wherein at least one neoepitope derived fromthe at least one neoantigenic peptide is presented on the surface of thetumor cell. In some embodiments, the neoepitope is presented on thesurface of the tumor cell in complex with a MHC class I molecule. Insome embodiments, the neoepitope is presented on the surface of thetumor cell in complex with a MHC class II molecule.

In some embodiments, a method comprises administering to a subject inneed thereof a therapeutically effective amount of a neoantigenicpolypeptide or polynucleotide described herein that delivers anexogenous polypeptide comprising at least one antigenic peptide to atumor cell, wherein the neoepitope is presented on the surface of thetumor cell, and an immune response against the tumor cell is induced. Insome embodiments, the immune response against the tumor cell isincreased. In some embodiments, the neoantigenic polypeptide orpolynucleotide delivers an exogenous polypeptide comprising at least oneneoantigenic peptide to a tumor cell, wherein the neoepitope ispresented on the surface of the tumor cell, and tumor growth isinhibited.

In some embodiments, a method comprises administering to a subject inneed thereof a therapeutically effective amount of a neoantigenicpolypeptide or polynucleotide described herein that delivers anexogenous polypeptide comprising at least one neoantigenic peptide to atumor cell, wherein the neoepitope derived from the at least oneneoantigenic peptide is presented on the surface of the tumor cell, andT cell killing directed against the tumor cell is induced. In someembodiments, T cell killing directed against the tumor cell is enhanced.In some embodiments, T cell killing directed against the tumor cell isincreased.

In some embodiments, a method of increasing an immune response in asubject comprises administering to the subject a therapeuticallyeffective amount of a neoantigenic therapeutic described herein, whereinthe agent is an antibody that specifically binds the neoantigendescribed herein. In some embodiments, a method of increasing an immuneresponse in a subject comprises administering to the subject atherapeutically effective amount of the antibody.

The present disclosure provides methods of redirecting an existingimmune response to a tumor. In some embodiments, a method of redirectingan existing immune response to a tumor comprises administering to asubject a therapeutically effective amount of a neoantigen therapeuticdescribed herein. In some embodiments, the existing immune response isagainst a virus. In some embodiments, the virus is selected from thegroup consisting of: measles virus, varicella-zoster virus (VZV;chickenpox virus), influenza virus, mumps virus, poliovirus, rubellavirus, rotavirus, hepatitis A virus (HAV), hepatitis B virus (HBV),Epstein Barr virus (EBV), and cytomegalovirus (CMV). In someembodiments, the virus is varicella-zoster virus. In some embodiments,the virus is cytomegalovirus. In some embodiments, the virus is measlesvirus. In some embodiments, the existing immune response has beenacquired after a natural viral infection. In some embodiments, theexisting immune response has been acquired after vaccination against avirus. In some embodiments, the existing immune response is acell-mediated response. In some embodiments, the existing immuneresponse comprises cytotoxic T cells (CTLs) or Th cells.

In some embodiments, a method of redirecting an existing immune responseto a tumor in a subject comprises administering a fusion proteincomprising (i) an antibody that specifically binds a neoantigen and (ii)at least one neoantigenic peptide described herein, wherein (a) thefusion protein is internalized by a tumor cell after binding to thetumor-associated antigen or the neoepitope; (b) the neoantigenic peptideis processed and presented on the surface of the tumor cell associatedwith a MHC class I molecule; and (c) the neoantigenic peptide/MHC ClassI complex is recognized by cytotoxic T cells. In some embodiments, thecytotoxic T cells are memory T cells. In some embodiments, the memory Tcells are the result of a vaccination with the neoantigenic peptide.

The present disclosure provides methods of increasing the immunogenicityof a tumor. In some embodiments, a method of increasing theimmunogenicity of a tumor comprises contacting a tumor or tumor cellswith an effective amount of a neoantigen therapeutic described herein.In some embodiments, a method of increasing the immunogenicity of atumor comprises administering to a subject a therapeutically effectiveamount of a neoantigen therapeutic described herein.

The present disclosure also provides methods for inhibiting growth of atumor using a neoantigen therapeutic described herein. In certainembodiments, a method of inhibiting growth of a tumor comprisescontacting a cell mixture with a neoantigen therapeutic in vitro. Forexample, an immortalized cell line or a cancer cell line mixed withimmune cells (e.g., T cells) is cultured in medium to which aneoantigenic peptide is added. In some embodiments, tumor cells areisolated from a patient sample, for example, a tissue biopsy, pleuraleffusion, or blood sample, mixed with immune cells (e.g., T cells), andcultured in medium to which a neoantigen therapeutic is added. In someembodiments, a neoantigen therapeutic increases, promotes, and/orenhances the activity of the immune cells. In some embodiments, aneoantigen therapeutic inhibits tumor cell growth. In some embodiments,a neoantigen therapeutic activates killing of the tumor cells.

In certain embodiments, the subject is a human. In certain embodiments,the subject has a tumor or the subject had a tumor which was at leastpartially removed.

In some embodiments, a method of inhibiting growth of a tumor comprisesredirecting an existing immune response to a new target, comprisingadministering to a subject a therapeutically effective amount of aneoantigen therapeutic, wherein the existing immune response is againstan antigenic peptide delivered to the tumor cell by the neoantigenicpeptide.

In certain embodiments, the tumor comprises cancer stem cells. Incertain embodiments, the frequency of cancer stem cells in the tumor isreduced by administration of the neoantigen therapeutic. In someembodiments, a method of reducing the frequency of cancer stem cells ina tumor in a subject, comprising administering to the subject atherapeutically effective amount of a neoantigen therapeutic isprovided.

In addition, in some aspects the present disclosure provides a method ofreducing the tumorigenicity of a tumor in a subject, comprisingadministering to the subject a therapeutically effective amount of aneoantigen therapeutic described herein. In certain embodiments, thetumor comprises cancer stem cells. In some embodiments, thetumorigenicity of a tumor is reduced by reducing the frequency of cancerstem cells in the tumor. In some embodiments, the methods comprise usingthe neoantigen therapeutic described herein. In certain embodiments, thefrequency of cancer stem cells in the tumor is reduced by administrationof a neoantigen therapeutic described herein.

In some embodiments, the tumor is a solid tumor. In certain embodiments,the tumor is a tumor selected from the group consisting of: colorectaltumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breasttumor, kidney tumor, prostate tumor, neuroendocrine tumor,gastrointestinal tumor, melanoma, cervical tumor, bladder tumor,glioblastoma, and head and neck tumor. In certain embodiments, the tumoris a colorectal tumor. In certain embodiments, the tumor is an ovariantumor. In some embodiments, the tumor is a breast tumor. In someembodiments, the tumor is a lung tumor. In certain embodiments, thetumor is a pancreatic tumor. In certain embodiments, the tumor is amelanoma tumor. In some embodiments, the tumor is a solid tumor.

The present disclosure further provides methods for treating cancer in asubject comprising administering to the subject a therapeuticallyeffective amount of a neoantigen therapeutic described herein.

In some embodiments, a method of treating cancer comprises redirectingan existing immune response to a new target, the method comprisingadministering to a subject a therapeutically effective amount ofneoantigen therapeutic, wherein the existing immune response is againstan antigenic peptide delivered to the cancer cell by the neoantigenicpeptide.

The present disclosure provides for methods of treating cancercomprising administering to a subject a therapeutically effective amountof a neoantigen therapeutic described herein (e.g., a subject in need oftreatment). In certain embodiments, the subject is a human. In certainembodiments, the subject has a cancerous tumor. In certain embodiments,the subject has had a tumor at least partially removed.

Subjects can be, for example, mammal, humans, pregnant women, elderlyadults, adults, adolescents, pre-adolescents, children, toddlers,infants, newborn, or neonates. A subject can be a patient. In somecases, a subject can be a human. In some cases, a subject can be a child(i.e. a young human being below the age of puberty). In some cases, asubject can be an infant. In some cases, the subject can be aformula-fed infant. In some cases, a subject can be an individualenrolled in a clinical study. In some cases, a subject can be alaboratory animal, for example, a mammal, or a rodent. In some cases,the subject can be a mouse. In some cases, the subject can be an obeseor overweight subject.

In some embodiments, the subject has previously been treated with one ormore different cancer treatment modalities. In some embodiments, thesubject has previously been treated with one or more of radiotherapy,chemotherapy, or immunotherapy. In some embodiments, the subject hasbeen treated with one, two, three, four, or five lines of prior therapy.In some embodiments, the prior therapy is a cytotoxic therapy.

In certain embodiments, the cancer is a cancer selected from the groupconsisting of colorectal cancer, pancreatic cancer, lung cancer, ovariancancer, liver cancer, breast cancer, kidney cancer, prostate cancer,gastrointestinal cancer, melanoma, cervical cancer, neuroendocrinecancer, bladder cancer, glioblastoma, and head and neck cancer. Incertain embodiments, the cancer is pancreatic cancer. In certainembodiments, the cancer is ovarian cancer. In certain embodiments, thecancer is colorectal cancer. In certain embodiments, the cancer isbreast cancer. In certain embodiments, the cancer is prostate cancer. Incertain embodiments, the cancer is lung cancer. In certain embodiments,the cancer is melanoma. In some embodiments, the cancer is a solidcancer. In some embodiments, the cancer comprises a solid tumor.

In some embodiments, the cancer is a hematologic cancer. In someembodiment, the cancer is selected from the group consisting of: acutemyelogenous leukemia (AML), Hodgkin lymphoma, multiple myeloma, T cellacute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia(CLL), hairy cell leukemia, chronic myelogenous leukemia (CML),non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle celllymphoma (MCL), and cutaneous T cell lymphoma (CTCL).

In some embodiments, the neoantigen therapeutic is administered as acombination therapy. Combination therapy with two or more therapeuticagents uses agents that work by different mechanisms of action, althoughthis is not required. Combination therapy using agents with differentmechanisms of action can result in additive or synergetic effects.Combination therapy can allow for a lower dose of each agent than isused in monotherapy, thereby reducing toxic side effects and/orincreasing the therapeutic index of the agent(s). Combination therapycan decrease the likelihood that resistant cancer cells will develop. Insome embodiments, combination therapy comprises a therapeutic agent thataffects the immune response (e.g., enhances or activates the response)and a therapeutic agent that affects (e.g., inhibits or kills) thetumor/cancer cells.

In some instances, an immunogenic pharmaceutical composition can beadministered with an additional agent. The choice of the additionalagent can depend, at least in part, on the condition being treated. Theadditional agent can include, for example, a checkpoint inhibitor agentsuch as an anti-PD1, anti-CTLA4, anti-PD-L1, anti CD40, or anti-TIM3agent (e.g., an anti-PD1, anti-CTLA4, anti-PD-L1, anti CD40, oranti-TIM3 antibody); or any agents having a therapeutic effect for apathogen infection (e.g. viral infection), including, e.g., drugs usedto treat inflammatory conditions such as an NSAID, e.g., ibuprofen,naproxen, acetaminophen, ketoprofen, or aspirin. For example, thecheckpoint inhibitor can be a PD-1/PD-L1 antagonist selected from thegroup consisting of: nivolumab (ONO-4538/BMS-936558, MDX1 106, OPDIVO),pembrolizumab (MK-3475, KEYTRUDA), pidilizumab (CT-011), and MPDL328OA(ROCHE). As another example, formulations can additionally contain oneor more supplements, such as vitamin C, E or other anti-oxidants.

The methods of the disclosure can be used to treat any type of cancerknown in the art. Non-limiting examples of cancers to be treated by themethods of the present disclosure can include melanoma (e.g., metastaticmalignant melanoma), renal cancer (e.g., clear cell carcinoma), prostatecancer (e.g., hormone refractory prostate adenocarcinoma), pancreaticadenocarcinoma, breast cancer, colon cancer, lung cancer (e.g.,non-small cell lung cancer), esophageal cancer, squamous cell carcinomaof the head and neck, liver cancer, ovarian cancer, cervical cancer,thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and otherneoplastic malignancies.

Additionally, the disease or condition provided herein includesrefractory or recurrent malignancies whose growth may be inhibited usingthe methods of treatment of the present disclosure. In some embodiments,a cancer to be treated by the methods of treatment of the presentdisclosure is selected from the group consisting of carcinoma, squamouscarcinoma, adenocarcinoma, sarcomata, endometrial cancer, breast cancer,ovarian cancer, cervical cancer, fallopian tube cancer, primaryperitoneal cancer, colon cancer, colorectal cancer, squamous cellcarcinoma of the anogenital region, melanoma, renal cell carcinoma, lungcancer, non-small cell lung cancer, squamous cell carcinoma of the lung,stomach cancer, bladder cancer, gall bladder cancer, liver cancer,thyroid cancer, laryngeal cancer, salivary gland cancer, esophagealcancer, head and neck cancer, glioblastoma, glioma, squamous cellcarcinoma of the head and neck, prostate cancer, pancreatic cancer,mesothelioma, sarcoma, hematological cancer, leukemia, lymphoma,neuroma, and combinations thereof. In some embodiments, a cancer to betreated by the methods of the present disclosure include, for example,carcinoma, squamous carcinoma (for example, cervical canal, eyelid,tunica conjunctiva, vagina, lung, oral cavity, skin, urinary bladder,tongue, larynx, and gullet), and adenocarcinoma (for example, prostate,small intestine, endometrium, cervical canal, large intestine, lung,pancreas, gullet, rectum, uterus, stomach, mammary gland, and ovary). Insome embodiments, a cancer to be treated by the methods of the presentdisclosure further include sarcomata (for example, myogenic sarcoma),leukosis, neuroma, melanoma, and lymphoma. In some embodiments, a cancerto be treated by the methods of the present disclosure is breast cancer.In some embodiments, a cancer to be treated by the methods of treatmentof the present disclosure is triple negative breast cancer (TNBC). Insome embodiments, a cancer to be treated by the methods of treatment ofthe present disclosure is ovarian cancer. In some embodiments, a cancerto be treated by the methods of treatment of the present disclosure iscolorectal cancer.

In some embodiments, a patient or population of patients to be treatedwith a pharmaceutical composition of the present disclosure have a solidtumor. In some embodiments, a solid tumor is a melanoma, renal cellcarcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer,colon cancer, gall bladder cancer, laryngeal cancer, liver cancer,thyroid cancer, stomach cancer, salivary gland cancer, prostate cancer,pancreatic cancer, or Merkel cell carcinoma. In some embodiments, apatient or population of patients to be treated with a pharmaceuticalcomposition of the present disclosure have a hematological cancer. Insome embodiments, the patient has a hematological cancer such as Diffuselarge B cell lymphoma (“DLBCL”), Hodgkin's lymphoma (“HL”),Non-Hodgkin's lymphoma (“NHL”), Follicular lymphoma (“FL”), acutemyeloid leukemia (“AML”), or Multiple myeloma (“MM”). In someembodiments, a patient or population of patients to be treated havingthe cancer selected from the group consisting of ovarian cancer, lungcancer and melanoma.

Specific examples of cancers that can be prevented and/or treated inaccordance with present disclosure include, but are not limited to, thefollowing: renal cancer, kidney cancer, glioblastoma multiforme,metastatic breast cancer; breast carcinoma; breast sarcoma;neurofibroma; neurofibromatosis; pediatric tumors; neuroblastoma;malignant melanoma; carcinomas of the epidermis; leukemias such as butnot limited to, acute leukemia, acute lymphocytic leukemia, acutemyelocytic leukemias such as myeloblastic, promyelocytic,myelomonocytic, monocytic, erythroleukemia leukemias and myclodysplasticsyndrome, chronic leukemias such as but not limited to, chronicmyelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairycell leukemia; polycythemia vera; lymphomas such as but not limited toHodgkin's disease, non-Hodgkin's disease; multiple myelomas such as butnot limited to smoldering multiple myeloma, nonsecretory myeloma,osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma andextramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonalgammopathy of undetermined significance; benign monoclonal gammopathy;heavy chain disease; bone cancer and connective tissue sarcomas such asbut not limited to bone sarcoma, myeloma bone disease, multiple myeloma,cholesteatoma-induced bone osteosarcoma, Paget's disease of bone,osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant celltumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissuesarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi'ssarcoma, leiomyosarcoma, liposarcoma, lymphangio sarcoma, neurilemmoma,rhabdomyosarcoma, and synovial sarcoma; brain tumors such as but notlimited to, glioma, astrocytoma, brain stem glioma, ependymoma,oligodendroglioma, nonglial tumor, acoustic neurinoma,craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, and primary brain lymphoma; breast cancer including butnot limited to adenocarcinoma, lobular (small cell) carcinoma,intraductal carcinoma, medullary breast cancer, mucinous breast cancer,tubular breast cancer, papillary breast cancer, Paget's disease(including juvenile Paget's disease) and inflammatory breast cancer;adrenal cancer such as but not limited to pheochromocytom andadrenocortical carcinoma; thyroid cancer such as but not limited topapillary or follicular thyroid cancer, medullary thyroid cancer andanaplastic thyroid cancer; pancreatic cancer such as but not limited to,insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secretingtumor, and carcinoid or islet cell tumor; pituitary cancers such as butlimited to Cushing's disease, prolactin-secreting tumor, acromegaly, anddiabetes insipius; eye cancers such as but not limited to ocularmelanoma such as iris melanoma, choroidal melanoma, and cilliary bodymelanoma, and retinoblastoma; vaginal cancers such as squamous cellcarcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamouscell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma,and Paget's disease; cervical cancers such as but not limited to,squamous cell carcinoma, and adenocarcinoma; uterine cancers such as butnot limited to endometrial carcinoma and uterine sarcoma; ovariancancers such as but not limited to, ovarian epithelial carcinoma,borderline tumor, germ cell tumor, and stromal tumor; cervicalcarcinoma; esophageal cancers such as but not limited to, squamouscancer, adenocarcinoma, adenoid cyctic carcinoma, mucoepidermoidcarcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma,verrucous carcinoma, and oat cell (small cell) carcinoma; stomachcancers such as but not limited to, adenocarcinoma, fungating(polypoid), ulcerating, superficial spreading, diffusely spreading,malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; coloncancers; colorectal cancer, KRAS mutated colorectal cancer; coloncarcinoma; rectal cancers; liver cancers such as but not limited tohepatocellular carcinoma and hepatoblastoma, gallbladder cancers such asadenocarcinoma; cholangiocarcinomas such as but not limited topappillary, nodular, and diffuse; lung cancers such as KRAS-mutatednon-small cell lung cancer, non-small cell lung cancer, squamous cellcarcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinomaand small-cell lung cancer; lung carcinoma; testicular cancers such asbut not limited to germinal tumor, seminoma, anaplastic, classic(typical), spermatocytic, nonseminoma, embryonal carcinoma, teratomacarcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers such asbut not limited to, androgen-independent prostate cancer,androgen-dependent prostate cancer, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers such as but not limited tosquamous cell carcinoma; basal cancers; salivary gland cancers such asbut not limited to adenocarcinoma, mucoepidermoid carcinoma, andadenoidcystic carcinoma; pharynx cancers such as but not limited tosquamous cell cancer, and verrucous; skin cancers such as but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acrallentiginous melanoma; kidney cancers such as but notlimited to renal cell cancer, adenocarcinoma, hypernephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);renal carcinoma; Wilms' tumor; bladder cancers such as but not limitedto transitional cell carcinoma, squamous cell cancer, adenocarcinoma,carcinosarcoma. In addition, cancers include myxosarcoma, osteogenicsarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma,synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma,bronchogenic carcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma and papillary adenocarcinomas.

Cancers include, but are not limited to, B cell cancer, e.g., multiplemyeloma, Waldenstrom's macroglobulinemia, the heavy chain diseases, suchas, for example, alpha chain disease, gamma chain disease, and mu chaindisease, benign monoclonal gammopathy, and immunocytic amyloidosis,melanomas, breast cancer, lung cancer, bronchus cancer, colorectalcancer, prostate cancer (e.g., metastatic, hormone refractory prostatecancer), pancreatic cancer, stomach cancer, ovarian cancer, urinarybladder cancer, brain or central nervous system cancer, peripheralnervous system cancer, esophageal cancer, cervical cancer, uterine orendometrial cancer, cancer of the oral cavity or pharynx, liver cancer,kidney cancer, testicular cancer, biliary tract cancer, small bowel orappendix cancer, salivary gland cancer, thyroid gland cancer, adrenalgland cancer, osteosarcoma, chondrosarcoma, cancer of hematologicaltissues, and the like. Other non-limiting examples of types of cancersapplicable to the methods encompassed by the present disclosure includehuman sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer,breast cancer, ovarian cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, liver cancer,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, bone cancer, brain tumor, testicular cancer, lung carcinoma,small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g.,acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic,promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronicleukemia (chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, and heavy chain disease. In some embodiments, thecancer whose phenotype is determined by the method of the presentdisclosure is an epithelial cancer such as, but not limited to, bladdercancer, breast cancer, cervical cancer, colon cancer, gynecologiccancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, headand neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, orskin cancer. In other embodiments, the cancer is breast cancer, prostatecancer, lung cancer, or colon cancer. In still other embodiments, theepithelial cancer is non-small-cell lung cancer, nonpapillary renal cellcarcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovariancarcinoma), or breast carcinoma. The epithelial cancers may becharacterized in various other ways including, but not limited to,serous, endometrioid, mucinous, clear cell, brenner, orundifferentiated. In some embodiments, the present disclosure is used inthe treatment, diagnosis, and/or prognosis of lymphoma or its subtypes,including, but not limited to, mantle cell lymphoma. Lymphoproliferativedisorders are also considered to be proliferative diseases.

In some embodiments, the combination of an agent described herein and atleast one additional therapeutic agent results in additive orsynergistic results. In some embodiments, the combination therapyresults in an increase in the therapeutic index of the agent. In someembodiments, the combination therapy results in an increase in thetherapeutic index of the additional therapeutic agent(s). In someembodiments, the combination therapy results in a decrease in thetoxicity and/or side effects of the agent. In some embodiments, thecombination therapy results in a decrease in the toxicity and/or sideeffects of the additional therapeutic agent(s).

In certain embodiments, in addition to administering a neoantigentherapeutic described herein, the method or treatment further comprisesadministering at least one additional therapeutic agent. An additionaltherapeutic agent can be administered prior to, concurrently with,and/or subsequently to, administration of the agent. In someembodiments, the at least one additional therapeutic agent comprises 1,2, 3, or more additional therapeutic agents.

Therapeutic agents that can be administered in combination with theneoantigen therapeutic described herein include chemotherapeutic agents.Thus, in some embodiments, the method or treatment involves theadministration of an agent described herein in combination with achemotherapeutic agent or in combination with a cocktail ofchemotherapeutic agents. Treatment with an agent can occur prior to,concurrently with, or subsequent to administration of chemotherapies.Combined administration can include co-administration, either in asingle pharmaceutical formulation or using separate formulations, orconsecutive administration in either order but generally within a timeperiod such that all active agents can exert their biological activitiessimultaneously. Preparation and dosing schedules for suchchemotherapeutic agents can be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for such chemotherapy are alsodescribed in The Chemotherapy Source Book, 4th Edition, 2008, M. C.Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, Pa.

Useful classes of chemotherapeutic agents include, for example,anti-tubulin agents, auristatins, DNA minor groove binders, DNAreplication inhibitors, alkylating agents (e.g., platinum complexes suchas cisplatin, mono(platinum), bis(platinum) and tri-nuclear platinumcomplexes and carboplatin), anthracyclines, antibiotics, anti-folates,antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides,fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas,platinols, purine antimetabolites, puromycins, radiation sensitizers,steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or thelike. In certain embodiments, the second therapeutic agent is analkylating agent, an antimetabolite, an antimitotic, a topoisomeraseinhibitor, or an angiogenesis inhibitor.

Chemotherapeutic agents useful in the present disclosure include, butare not limited to, alkylating agents such as thiotepa andcyclosphosphamide (CYTOXAN); alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamime; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine,floxuridine, 5-FU; androgens such as calusterone, dromostanolonepropionate, epitiostanol, mepitiostane, testolactone; anti-adrenals suchas aminoglutethimide, mitotane, trilostane; folic acid replenishers suchas folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (Ara-C); taxoids, e.g. paclitaxel (TAXOL) and docetaxel(TAXOTERE); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; ibandronate; CPT11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine (XELODA); and pharmaceutically acceptable salts, acids orderivatives of any of the above. Chemotherapeutic agents also includeanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON);and anti-androgens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above. In certain embodiments, theadditional therapeutic agent is cisplatin. In certain embodiments, theadditional therapeutic agent is carboplatin.

In certain embodiments, the chemotherapeutic agent is a topoisomeraseinhibitor. Topoisomerase inhibitors are chemotherapy agents thatinterfere with the action of a topoisomerase enzyme (e.g., topoisomeraseI or II). Topoisomerase inhibitors include, but are not limited to,doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D,etoposide, topotecan HCl, teniposide (VM-26), and irinotecan, as well aspharmaceutically acceptable salts, acids, or derivatives of any ofthese. In some embodiments, the additional therapeutic agent isirinotecan.

In certain embodiments, the chemotherapeutic agent is ananti-metabolite. An anti-metabolite is a chemical with a structure thatis similar to a metabolite required for normal biochemical reactions,yet different enough to interfere with one or more normal functions ofcells, such as cell division. Anti-metabolites include, but are notlimited to, gemcitabine, fluorouracil, capecitabine, methotrexatesodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside,thioguanine, 5-azacytidine, 6 mercaptopurine, azathioprine,6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, aswell as pharmaceutically acceptable salts, acids, or derivatives of anyof these. In certain embodiments, the additional therapeutic agent isgemcitabine.

In certain embodiments, the chemotherapeutic agent is an antimitoticagent, including, but not limited to, agents that bind tubulin. In someembodiments, the agent is a taxane. In certain embodiments, the agent ispaclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, orderivative of paclitaxel or docetaxel. In certain embodiments, the agentis paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel(ABRAXANE), DHA-paclitaxel, or PG-paclitaxel. In certain alternativeembodiments, the antimitotic agent comprises a vinca alkaloid, such asvincristine, vinblastine, vinorelbine, or vindesine, or pharmaceuticallyacceptable salts, acids, or derivatives thereof. In some embodiments,the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor ofa mitotic kinase such as Aurora A or Plk1. In certain embodiments, theadditional therapeutic agent is paclitaxel. In some embodiments, theadditional therapeutic agent is albumin-bound paclitaxel.

In some embodiments, an additional therapeutic agent comprises an agentsuch as a small molecule. For example, treatment can involve thecombined administration of an agent of the present disclosure with asmall molecule that acts as an inhibitor against tumor-associatedantigens including, but not limited to, EGFR, HER2 (ErbB2), and/or VEGF.In some embodiments, an agent of the present disclosure is administeredin combination with a protein kinase inhibitor selected from the groupconsisting of: gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib(SUTENT), lapatanib, vandetanib (ZACTIMA), AEE788, CI-1033, cediranib(RECENTIN), sorafenib (NEXAVAR), and pazopanib (GW786034B). In someembodiments, an additional therapeutic agent comprises an mTORinhibitor. In another embodiment, the additional therapeutic agent ischemotherapy or other inhibitors that reduce the number of Treg cells.In certain embodiments, the therapeutic agent is cyclophosphamide or ananti-CTLA4 antibody. In another embodiment, the additional therapeuticreduces the presence of myeloid-derived suppressor cells. In a furtherembodiment, the additional therapeutic is carbotaxol. In anotherembodiment, the additional therapeutic agent shifts cells to a T helper1 response. In a further embodiment, the additional therapeutic agent isibrutinib.

In some embodiments, an additional therapeutic agent comprises abiological molecule, such as an antibody. For example, treatment caninvolve the combined administration of an agent of the presentdisclosure with antibodies against tumor-associated antigens including,but not limited to, antibodies that bind EGFR, HER2/ErbB2, and/or VEGF.In certain embodiments, the additional therapeutic agent is an antibodyspecific for a cancer stem cell marker. In certain embodiments, theadditional therapeutic agent is an antibody that is an angiogenesisinhibitor (e.g., an anti-VEGF or VEGF receptor antibody). In certainembodiments, the additional therapeutic agent is bevacizumab (AVASTIN),ramucirumab, trastuzumab (HERCEPTIN), pertuzumab (OMNITARG), panitumumab(VECTIBIX), nimotuzumab, zalutumumab, or cetuximab (ERBITUX).

The agents and compositions provided herein may be used alone or incombination with conventional therapeutic regimens such as surgery,irradiation, chemotherapy and/or bone marrow transplantation(autologous, syngeneic, allogeneic or unrelated). A set of tumorantigens can be useful, e.g., in a large fraction of cancer patients.

In some embodiments, at least one or more chemotherapeutic agents may beadministered in addition to the composition comprising an immunogenicvaccine. In some embodiments, the one or more chemotherapeutic agentsmay belong to different classes of chemotherapeutic agents.

Examples of chemotherapy agents include, but are not limited to,alkylating agents such as nitrogen mustards (e.g. mechlorethamine(nitrogen mustard), chlorambucil, cyclophosphamide (Cytoxan®),ifosfamide, and melphalan); nitrosoureas (e.g. N-Nitroso-N-methylurea,streptozocin, carmustine (BCNU), lomustine, and semustine); alkylsulfonates (e.g. busulfan); tetrazines (e.g. dacarbazine (DTIC),mitozolomide and temozolomide (Temodar®)); aziridines (e.g. thiotepa,mytomycin and diaziquone); and platinum drugs (e.g. cisplatin,carboplatin, and oxaliplatin); non-classical alkylating agents such asprocarbazine and altretamine (hexamethylmelamine); anti-metaboliteagents such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP),capecitabine (Xeloda®), cladribine, clofarabine, cytarabine (Ara-C®),decitabine, floxuridine, fludarabine, nelarabine, gemcitabine (Gemzar®),hydroxyurea, methotrexate, pemetrexed (Alimta®), pentostatin,thioguanine, Vidaza; anti-microtubule agents such as vinca alkaloids(e.g. vincristine, vinblastine, vinorelbine, vindesine and vinflunine);taxanes (e.g. paclitaxel (Taxol®), docetaxel (Taxotere®));podophyllotoxin (e.g. etoposide and teniposide); epothilones (e.g.ixabepilone (Ixempra®)); estramustine (Emcyt®); anti-tumor antibioticssuch as anthracyclines (e.g. daunorubicin, doxorubicin (Adriamycin®,epirubicin, idarubicin); actinomycin-D; and bleomycin; topoisomerase Iinhibitors such as topotecan and irinotecan (CPT-11); topoisomerase IIinhibitors such as etoposide (VP-16), teniposide, mitoxantrone,novobiocin, merbarone and aclarubicin; corticosteroids such asprednisone, methylprednisolone (Solumedrol®), and dexamethasone(Decadron®); L-asparaginase; bortezomib (Velcade®); immunotherapeuticagents such as rituximab (Rituxan®), alemtuzumab (Campath®),thalidomide, lenalidomide (Revlimid®), BCG, interleukin-2,interferon-alfa and cancer vaccines such as Provenge®; hormonetherapeutic agents such as fulvestrant (Faslodex®), tamoxifen,toremifene (Fareston®), anastrozole (Arimidex®), exemestan (Aromasin®),letrozole (Ferrara®), megestrol acetate (Megace®), estrogens,bicalutamide (Casodex®), flutamide (Eulexin®), nilutamide (Nilandron®),leuprolide (Lupron®) and goserelin (Zoladex®); differentiating agentssuch as retinoids, tretinoin (ATRA or Atralin®), bexarotene (Targretin®)and arsenic trioxide (Arsenox®); and targeted therapeutic agents such asimatinib (Gleevec®), gefitinib (Iressa®) and sunitinib (Sutent®). Insome embodiments, the chemotherapy is a cocktail therapy. Examples of acocktail therapy includes, but is not limited to, CHOP/R-CHOP (rituxan,cyclophosphamide, hydroxydoxorubicin, vincristine, and prednisone),EPOCH (etoposide, prednisone, vincristine, cyclophosphamide,hydroxydoxorubicin), Hyper-CVAD (cyclophosphamide, vincristine,hydroxydoxorubicin, dexamethasone), FOLFOX (fluorouracil (5-FU),leucovorin, oxaliplatin), ICE (ifosfamide, carboplatin, etoposide), DHAP(high-dose cytarabine [ara-C], dexamethasone, cisplatin), ESHAP(etoposide, methylprednisolone, cytarabine [ara-C], cisplatin) and CMF(cyclophosphamide, methotrexate, fluouracil).

In certain embodiments, an additional therapeutic agent comprises asecond immunotherapeutic agent. In some embodiments, the additionalimmunotherapeutic agent includes, but is not limited to, a colonystimulating factor, an interleukin, an antibody that blocksimmunosuppressive functions (e.g., an anti-CTLA-4 antibody, anti-CD28antibody, anti-CD3 antibody, anti-PD-1 antibody, anti-PD-L1 antibody,anti-TIGIT antibody), an antibody that enhances immune cell functions(e.g., an anti-GITR antibody, an anti-OX-40 antibody, an anti-CD40antibody, or an anti-4-IBB antibody), a toll-like receptor (e.g., TLR4,TLR7, TLR9), a soluble ligand (e.g., GITRL, GITRL-Fc, OX-40L, OX-40L-Fc,CD40L, CD40L-Fc, 4-1BB ligand, or 4-1BB ligand-Fc), or a member of theB7 family (e.g., CD80, CD86). In some embodiments, the additionalimmunotherapeutic agent targets CTLA-4, CD28, CD3, PD-1, PD-L1, TIGIT,GITR, OX-40, CD-40, or 4-IBB.

In some embodiments, the additional therapeutic agent is an immunecheckpoint inhibitor. In some embodiments, the immune checkpointinhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, ananti-CTLA-4 antibody, an anti-CD28 antibody, an anti-TIGIT antibody, ananti-LAG3 antibody, an anti-TIM3 antibody, an anti-GITR antibody, ananti-4-IBB antibody, or an anti-OX-40 antibody. In some embodiments, theadditional therapeutic agent is an anti-TIGIT antibody. In someembodiments, the additional therapeutic agent is an anti-PD-1 antibodyselected from the group consisting of: nivolumab (OPDIVO), pembrolizumab(KEYTRUDA), pidilzumab, MEDI0680, REGN2810, BGB-A317, and PDR001. Insome embodiments, the additional therapeutic agent is an anti-PD-L1antibody selected from the group consisting of: BMS935559 (MDX-1105),atexolizumab (MPDL3280A), durvalumab (MEDI4736), and avelumab(MSB0010718C). In some embodiments, the additional therapeutic agent isan anti-CTLA-4 antibody selected from the group consisting of:ipilimumab (YERVOY) and tremelimumab. In some embodiments, theadditional therapeutic agent is an anti-LAG-3 antibody selected from thegroup consisting of: BMS-986016 and LAG525. In some embodiments, theadditional therapeutic agent is an anti-OX-40 antibody selected from thegroup consisting of: MEDI6469, MEDI0562, and MOXR0916. In someembodiments, the additional therapeutic agent is an anti-4-IBB antibodyselected from the group consisting of: PF-05082566.

In some embodiments, the neoantigen therapeutic can be administered incombination with a biologic molecule selected from the group consistingof: adrenomedullin (AM), angiopoietin (Ang), BMPs, BDNF, EGF,erythropoietin (EPO), FGF, GDNF, granulocyte colony stimulating factor(G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF),FLT-3L, macrophage colony stimulating factor (M-CSF), stem cell factor(SCF), GDF9, HGF, HDGF, IGF, migration-stimulating factor, myostatin(GDF-8), NGF, neurotrophins, PDGF, thrombopoietin, TGF-α, TGF-β, TNF-α,VEGF, P1GF, gamma-IFN, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12,IL-15, and IL-18.

In some embodiments, treatment with a neoantigen therapeutic describedherein can be accompanied by surgical removal of tumors, removal ofcancer cells, or any other surgical therapy deemed necessary by atreating physician.

In certain embodiments, treatment involves the administration of aneoantigen therapeutic described herein in combination with radiationtherapy. Treatment with an agent can occur prior to, concurrently with,or subsequent to administration of radiation therapy. Dosing schedulesfor such radiation therapy can be determined by the skilled medicalpractitioner.

Combined administration can include co-administration, either in asingle pharmaceutical formulation or using separate formulations, orconsecutive administration in either order but generally within a timeperiod such that all active agents can exert their biological activitiessimultaneously.

It will be appreciated that the combination of a neoantigen therapeuticdescribed herein and at least one additional therapeutic agent can beadministered in any order or concurrently. In some embodiments, theagent will be administered to patients that have previously undergonetreatment with a second therapeutic agent. In certain other embodiments,the neoantigen therapeutic and a second therapeutic agent will beadministered substantially simultaneously or concurrently. For example,a subject can be given an agent while undergoing a course of treatmentwith a second therapeutic agent (e.g., chemotherapy). In certainembodiments, a neoantigen therapeutic will be administered within 1 yearof the treatment with a second therapeutic agent. It will further beappreciated that the two (or more) agents or treatments can beadministered to the subject within a matter of hours or minutes (i.e.,substantially simultaneously).

For the treatment of a disease, the appropriate dosage of a neoantigentherapeutic described herein depends on the type of disease to betreated, the severity and course of the disease, the responsiveness ofthe disease, whether the agent is administered for therapeutic orpreventative purposes, previous therapy, the patient's clinical history,and so on, all at the discretion of the treating physician. Theneoantigen therapeutic can be administered one time or over a series oftreatments lasting from several days to several months, or until a cureis effected or a diminution of the disease state is achieved (e.g.,reduction in tumor size). Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient andwill vary depending on the relative potency of an individual agent. Theadministering physician can determine optimum dosages, dosingmethodologies, and repetition rates.

In some embodiments, a neoantigen therapeutic can be administered at aninitial higher “loading” dose, followed by one or more lower doses. Insome embodiments, the frequency of administration can also change. Insome embodiments, a dosing regimen can comprise administering an initialdose, followed by additional doses (or “maintenance” doses) once a week,once every two weeks, once every three weeks, or once every month. Forexample, a dosing regimen can comprise administering an initial loadingdose, followed by a weekly maintenance dose of, for example, one-half ofthe initial dose. Or a dosing regimen can comprise administering aninitial loading dose, followed by maintenance doses of, for exampleone-half of the initial dose every other week. Or a dosing regimen cancomprise administering three initial doses for 3 weeks, followed bymaintenance doses of, for example, the same amount every other week.

As is known to those of skill in the art, administration of anytherapeutic agent can lead to side effects and/or toxicities. In somecases, the side effects and/or toxicities are so severe as to precludeadministration of the particular agent at a therapeutically effectivedose. In some cases, therapy must be discontinued, and other agents canbe tried. However, many agents in the same therapeutic class displaysimilar side effects and/or toxicities, meaning that the patient eitherhas to stop therapy, or if possible, suffer from the unpleasant sideeffects associated with the therapeutic agent.

In some embodiments, the dosing schedule can be limited to a specificnumber of administrations or “cycles”. In some embodiments, the agent isadministered for 3, 4, 5, 6, 7, 8, or more cycles. For example, theagent is administered every 2 weeks for 6 cycles, the agent isadministered every 3 weeks for 6 cycles, the agent is administered every2 weeks for 4 cycles, the agent is administered every 3 weeks for 4cycles, etc. Dosing schedules can be decided upon and subsequentlymodified by those skilled in the art.

The present disclosure provides methods of administering to a subject aneoantigen therapeutic described herein comprising using an intermittentdosing strategy for administering one or more agents, which can reduceside effects and/or toxicities associated with administration of anagent, chemotherapeutic agent, etc. In some embodiments, a method fortreating cancer in a human subject comprises administering to thesubject a therapeutically effective dose of a neoantigen therapeutic incombination with a therapeutically effective dose of a chemotherapeuticagent, wherein one or both of the agents are administered according toan intermittent dosing strategy. In some embodiments, a method fortreating cancer in a human subject comprises administering to thesubject a therapeutically effective dose of a neoantigen therapeutic incombination with a therapeutically effective dose of a secondimmunotherapeutic agent, wherein one or both of the agents areadministered according to an intermittent dosing strategy. In someembodiments, the intermittent dosing strategy comprises administering aninitial dose of a neoantigen therapeutic to the subject, andadministering subsequent doses of the agent about once every 2 weeks. Insome embodiments, the intermittent dosing strategy comprisesadministering an initial dose of a neoantigen therapeutic to thesubject, and administering subsequent doses of the agent about onceevery 3 weeks. In some embodiments, the intermittent dosing strategycomprises administering an initial dose of a neoantigen therapeutic tothe subject, and administering subsequent doses of the agent about onceevery 4 weeks. In some embodiments, the agent is administered using anintermittent dosing strategy and the additional therapeutic agent isadministered weekly.

The present disclosure provides compositions comprising the neoantigentherapeutic described herein. The present disclosure also providespharmaceutical compositions comprising a neoantigen therapeuticdescribed herein and a pharmaceutically acceptable vehicle. In someembodiments, the pharmaceutical compositions find use in immunotherapy.In some embodiments, the compositions find use in inhibiting tumorgrowth. In some embodiments, the pharmaceutical compositions find use ininhibiting tumor growth in a subject (e.g., a human patient). In someembodiments, the compositions find use in treating cancer. In someembodiments, the pharmaceutical compositions find use in treating cancerin a subject (e.g., a human patient).

Formulations are prepared for storage and use by combining a neoantigentherapeutic of the present disclosure with a pharmaceutically acceptablevehicle (e.g., a carrier or excipient). Those of skill in the artgenerally consider pharmaceutically acceptable carriers, excipients,and/or stabilizers to be inactive ingredients of a formulation orpharmaceutical composition. Exemplary formulations are listed in WO2015/095811.

Suitable pharmaceutically acceptable vehicles include, but are notlimited to, nontoxic buffers such as phosphate, citrate, and otherorganic acids; salts such as sodium chloride; antioxidants includingascorbic acid and methionine; preservatives such asoctadecyldimethylbenzyl ammonium chloride, hexamethonium chloride,benzalkonium chloride, benzethonium chloride, phenol, butyl or benzylalcohol, alkyl parabens, such as methyl or propyl paraben, catechol,resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weightpolypeptides (e.g., less than about 10 amino acid residues); proteinssuch as serum albumin, gelatin, or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; carbohydrates such asmonosaccharides, disaccharides, glucose, mannose, or dextrins; chelatingagents such as EDTA; sugars such as sucrose, mannitol, trehalose orsorbitol; salt-forming counter-ions such as sodium; metal complexes suchas Zn-protein complexes; and non-ionic surfactants such as TWEEN orpolyethylene glycol (PEG). (Remington: The Science and Practice ofPharmacy, 22st Edition, 2012, Pharmaceutical Press, London.). In someembodiments, the vehicle is 5% dextrose in water.

The pharmaceutical compositions described herein can be administered inany number of ways for either local or systemic treatment.Administration can be topical by epidermal or transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders; pulmonary by inhalation or insufflation of powders oraerosols, including by nebulizer, intratracheal, and intranasal; oral;or parenteral including intravenous, intra-arterial, intratumoral,subcutaneous, intraperitoneal, intramuscular (e.g., injection orinfusion), or intracranial (e.g., intrathecal or intraventricular).

The therapeutic formulation can be in unit dosage form. Suchformulations include tablets, pills, capsules, powders, granules,solutions or suspensions in water or non-aqueous media, orsuppositories.

The neoantigenic peptides described herein can also be entrapped inmicrocapsules. Such microcapsules are prepared, for example, bycoacervation techniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nanoparticles and nanocapsules) or in macroemulsions asdescribed in Remington: The Science and Practice of Pharmacy, 22stEdition, 2012, Pharmaceutical Press, London.

In certain embodiments, pharmaceutical formulations include a neoantigentherapeutic described herein complexed with liposomes. Methods toproduce liposomes are known to those of skill in the art. For example,some liposomes can be generated by reverse phase evaporation with alipid composition comprising phosphatidylcholine, cholesterol, andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can beextruded through filters of defined pore size to yield liposomes withthe desired diameter.

In certain embodiments, sustained-release preparations comprising theneoantigenic peptides described herein can be produced. Suitableexamples of sustained-release preparations include semi-permeablematrices of solid hydrophobic polymers containing an agent, where thematrices are in the form of shaped articles (e.g., films ormicrocapsules). Examples of sustained-release matrices includepolyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) orpoly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(−)-3-hydroxybutyric acid.

The present disclosure provides methods of treatment comprising animmunogenic vaccine. Methods of treatment for a disease (such as canceror a viral infection) are provided. A method can comprise administeringto a subject an effective amount of a composition comprising animmunogenic antigen. In some embodiments, the antigen comprises a viralantigen. In some embodiments, the antigen comprises a tumor antigen.

Non-limiting examples of vaccines that can be prepared include apeptide-based vaccine, a nucleic acid-based vaccine, an antibody basedvaccine, a T cell based vaccine, and an antigen-presenting cell basedvaccine.

Vaccine compositions can be formulated using one or more physiologicallyacceptable carriers including excipients and auxiliaries whichfacilitate processing of the active agents into preparations which canbe used pharmaceutically. Proper formulation can be dependent upon theroute of administration chosen. Any of the well-known techniques,carriers, and excipients can be used as suitable and as understood inthe art.

In some cases, the vaccine composition is formulated as a peptide-basedvaccine, a nucleic acid-based vaccine, an antibody based vaccine, or acell based vaccine. For example, a vaccine composition can include nakedcDNA in cationic lipid formulations; lipopeptides (e.g., Vitiello, A. etal., J. Clin. Invest. 95:341, 1995), naked cDNA or peptides,encapsulated e.g., in poly(DL-lactide-co-glycolide) (“PLG”) microspheres(see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonsoet al, Vaccine 12:299-306, 1994; Jones et al, Vaccine 13:675-681, 1995);peptide composition contained in immune stimulating complexes (ISCOMS)(e.g., Takahashi et al, Nature 344:873-875, 1990; Hu et al, Clin. Exp.Immunol. 113:235-243, 1998); or multiple antigen peptide systems (MAPs)(see e.g., Tam, J. P., Proc. Natl Acad. Sci. U.S.A. 85:5409-5413, 1988;Tarn, J. P., J. Immunol. Methods 196:17-32, 1996). Sometimes, a vaccineis formulated as a peptide-based vaccine, or nucleic acid based vaccinein which the nucleic acid encodes the polypeptides. Sometimes, a vaccineis formulated as an antibody based vaccine. Sometimes, a vaccine isformulated as a cell based vaccine.

The amino acid sequence of an identified disease-specific immunogenicneoantigen peptide can be used develop a pharmaceutically acceptablecomposition. The source of antigen can be, but is not limited to,natural or synthetic proteins, including glycoproteins, peptides, andsuperantigens; antibody/antigen complexes; lipoproteins; RNA or atranslation product thereof; and DNA or a polypeptide encoded by theDNA. The source of antigen may also comprise non-transformed,transformed, transfected, or transduced cells or cell lines. Cells maybe transformed, transfected, or transduced using any of a variety ofexpression or retroviral vectors known to those of ordinary skill in theart that may be employed to express recombinant antigens. Expression mayalso be achieved in any appropriate host cell that has been transformed,transfected, or transduced with an expression or retroviral vectorcontaining a DNA molecule encoding recombinant antigen(s). Any number oftransfection, transformation, and transduction protocols known to thosein the art may be used. Recombinant vaccinia vectors and cells infectedwith the vaccinia vector, may be used as a source of antigen.

A composition can comprise a synthetic disease-specific immunogenicneoantigen peptide. A composition can comprise two or moredisease-specific immunogenic neoantigen peptides. A composition maycomprise a precursor to a disease-specific immunogenic peptide (such asa protein, peptide, DNA and RNA). A precursor to a disease-specificimmunogenic peptide can generate or be generated to the identifieddisease-specific immunogenic neoantigen peptide. In some embodiments, atherapeutic composition comprises a precursor of an immunogenic peptide.The precursor to a disease-specific immunogenic peptide can be apro-drug. In some embodiments, the composition comprising adisease-specific immunogenic neoantigen peptide may further comprise anadjuvant. For example, the neoantigen peptide can be utilized as avaccine. In some embodiments, an immunogenic vaccine may comprise apharmaceutically acceptable immunogenic neoantigen peptide. In someembodiments, an immunogenic vaccine may comprise a pharmaceuticallyacceptable precursor to an immunogenic neoantigen peptide (such as aprotein, peptide, DNA and RNA). In some embodiments, a method oftreatment comprises administering to a subject an effective amount of anantibody specifically recognizing an immunogenic neoantigen peptide. Insome embodiments, a method of treatment comprises administering to asubject an effective amount of a soluble TCR or TCR analog specificallyrecognizing an immunogenic neoantigen peptide.

The methods described herein are particularly useful in the personalizedmedicine context, where immunogenic neoantigen peptides are used todevelop therapeutics (such as vaccines or therapeutic antibodies) forthe same individual. Thus, a method of treating a disease in a subjectcan comprise identifying an immunogenic neoantigen peptide in a subjectaccording to the methods described herein; and synthesizing the peptide(or a precursor thereof); and administering the peptide or an antibodyspecifically recognizing the peptide to the subject. In someembodiments, an expression pattern of an immunogenic neoantigen canserve as the essential basis for the generation of patient specificvaccines. In some embodiments, an expression pattern of an immunogenicneoantigen can serve as the essential basis for the generation of avaccine for a group of patients with a particular disease. Thus,particular diseases, e.g., particular types of tumors, can beselectively treated in a patient group.

In some embodiments, the peptides described herein are structurallynormal antigens that can be recognized by autologous anti-disease Tcells in a large patient group. In some embodiments, anantigen-expression pattern of a group of diseased subjects whose diseaseexpresses structurally normal neoantigens is determined.

In some embodiments, the peptides described herein comprises a firstpeptide comprising a first neoepitope of a protein and a second peptidecomprising a second neoepitope of the same protein, wherein the firstpeptide is different from the second peptide, and wherein the firstneoepitope comprises a mutation and the second neoepitope comprises thesame mutation. In some embodiments, the peptides described hereincomprises a first peptide comprising a first neoepitope of a firstregion of a protein and a second peptide comprising a second neoepitopeof a second region of the same protein, wherein the first regioncomprises at least one amino acid of the second region, wherein thefirst peptide is different from the second peptide and wherein the firstneoepitope comprises a first mutation and the second neoepitopecomprises a second mutation. In some embodiments, the first mutation andthe second mutation are the same. In some embodiments, the mutation isselected from the group consisting of a point mutation, a splice-sitemutation, a frameshift mutation, a read-through mutation, a gene fusionmutation and any combination thereof.

There are a variety of ways in which to produce immunogenic neoantigens.Proteins or peptides may be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, in vitro translation, orthe chemical synthesis of proteins or peptides. In general, such diseasespecific neoantigens may be produced either in vitro or in vivo.Immunogenic neoantigens may be produced in vitro as peptides orpolypeptides, which may then be formulated into a personalized vaccineor immunogenic composition and administered to a subject. In vitroproduction of immunogenic neoantigens can comprise peptide synthesis orexpression of a peptide/polypeptide from a DNA or RNA molecule in any ofa variety of bacterial, eukaryotic, or viral recombinant expressionsystems, followed by purification of the expressed peptide/polypeptide.Alternatively, immunogenic neoantigens can be produced in vivo byintroducing molecules (e.g., DNA, RNA, and viral expression systems)that encode an immunogenic neoantigen into a subject, whereupon theencoded immunogenic neoantigens are expressed. In some embodiments, apolynucleotide encoding an immunogenic neoantigen peptide can be used toproduce the neoantigen peptide in vitro.

In some embodiments, a polynucleotide comprises a sequence with at least60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to a polynucleotide encoding an immunogenicneoantigen.

The polynucleotide may be, e.g., DNA, cDNA, PNA, CNA, RNA, single-and/or double-stranded, native or stabilized forms of polynucleotides,or combinations thereof. A nucleic acid encoding an immunogenicneoantigen peptide may or may not contain introns so long as it codesfor the peptide. In some embodiments in vitro translation is used toproduce the peptide.

Expression vectors comprising sequences encoding the neoantigen, as wellas host cells containing the expression vectors, are also contemplated.Expression vectors suitable for use in the present disclosure cancomprise at least one expression control element operationally linked tothe nucleic acid sequence. The expression control elements are insertedin the vector to control and regulate the expression of the nucleic acidsequence. Examples of expression control elements are well known in theart and include, for example, the lac system, operator and promoterregions of phage lambda, yeast promoters and promoters derived frompolyoma, adenovirus, retrovirus or SV40. Additional operational elementsinclude, but are not limited to, leader sequences, termination codons,polyadenylation signals and any other sequences necessary or preferredfor the appropriate transcription and subsequent translation of thenucleic acid sequence in the host system. It will be understood by oneskilled in the art the correct combination of expression controlelements will depend on the host system chosen. It will further beunderstood that the expression vector should contain additional elementsnecessary for the transfer and subsequent replication of the expressionvector containing the nucleic acid sequence in the host system. Examplesof such elements include, but are not limited to, origins of replicationand selectable markers.

The neoantigen peptides may be provided in the form of RNA or cDNAmolecules encoding the desired neoantigen peptides. One or moreneoantigen peptides of the present disclosure may be encoded by a singleexpression vector. Generally, the DNA is inserted into an expressionvector, such as a plasmid, in proper orientation and correct readingframe for expression, if necessary, the DNA may be linked to theappropriate transcriptional and translational regulatory controlnucleotide sequences recognized by the desired host (e.g., bacteria),although such controls are generally available in the expression vector.The vector is then introduced into the host bacteria for cloning usingstandard techniques. Useful expression vectors for eukaryotic hosts,especially mammals or humans include, for example, vectors comprisingexpression control sequences from SV40, bovine papilloma virus,adenovirus and cytomegalovirus. Useful expression vectors for bacterialhosts include known bacterial plasmids, such as plasmids from E. coli,including pCR 1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

In embodiments, a DNA sequence encoding a polypeptide of interest can beconstructed by chemical synthesis using an oligonucleotide synthesizer.Such oligonucleotides can be designed based on the amino acid sequenceof the desired polypeptide and selecting those codons that are favoredin the host cell in which the recombinant polypeptide of interest isproduced. Standard methods can be applied to synthesize an isolatedpolynucleotide sequence encoding an isolated polypeptide of interest.

Suitable host cells for expression of a polypeptide include prokaryotes,yeast, insect or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or bacilli. Higher eukaryoticcells include established cell lines of mammalian origin. Cell-freetranslation systems can also be employed. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are well known in the art. Various mammalian or insectcell culture systems can be employed to express recombinant protein.Exemplary mammalian host cell lines include, but are not limited toCOS-7, L cells, C127, 3T3, Chinese hamster ovary (CHO), 293, HeLa andBHK cell lines. Mammalian expression vectors can comprise nontranscribedelements such as an origin of replication, a suitable promoter andenhancer linked to the gene to be expressed, and other 5′ or 3′ flankingnontranscribed sequences, and 5′ or 3′ nontranslated sequences, such asnecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, and transcriptional termination sequences.

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography, and the like),centrifugation, differential solubility, or by any other standardtechnique for protein purification Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence,glutathione-S-transferase, and the like can be attached to the proteinto allow easy purification by passage over an appropriate affinitycolumn. Isolated proteins can also be physically characterized usingsuch techniques as proteolysis, nuclear magnetic resonance and x-raycrystallography.

A vaccine can comprise an entity that binds a polypeptide sequencedescribed herein. The entity can be an antibody. Antibody-based vaccinecan be formulated using any of the well-known techniques, carriers, andexcipients as suitable and as understood in the art. In someembodiments, the peptides described herein can be used for makingneoantigen specific therapeutics such as antibody therapeutics. Forexample, neoantigens can be used to raise and/or identify antibodiesspecifically recognizing the neoantigens. These antibodies can be usedas therapeutics. The antibody can be a natural antibody, a chimericantibody, a humanized antibody, or can be an antibody fragment. Theantibody may recognize one or more of the polypeptides described herein.In some embodiments, the antibody can recognize a polypeptide that has asequence with at most 40%, 50%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to a polypeptide describedherein. In some embodiments, the antibody can recognize a polypeptidethat has a sequence with at least 40%, 50%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to apolypeptide described herein. In some embodiments, the antibody canrecognize a polypeptide sequence that is at least 30%, 40%, 50%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of alength of a polypeptide described herein. In some embodiments, theantibody can recognize a polypeptide sequence that is at most 30%, 40%,50%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%of a length of a polypeptide described herein.

The present disclosure also contemplates the use of nucleic acidmolecules as vehicles for delivering neoantigen peptides/polypeptides tothe subject in need thereof, in vivo, in the form of, e.g., DNA/RNAvaccines.

In some embodiments, the vaccine is a nucleic acid vaccine. In someembodiments, neoantigens can be administered to a subject by use of aplasmid. Plasmids may be introduced into animal tissues by a number ofdifferent methods, e.g., injection or aerosol instillation of naked DNAon mucosal surfaces, such as the nasal and lung mucosa. In someembodiments, physical delivery, such as with a “gene-gun” may be used.The exact choice of expression vectors can depend upon thepeptide/polypeptides to be expressed, and is well within the skill ofthe ordinary artisan.

In some embodiments, the nucleic acid encodes an immunogenic peptide orpeptide precursor. In some embodiments, the nucleic acid vaccinecomprises sequences flanking the sequence coding the immunogenic peptideor peptide precursor. In some embodiments, the nucleic acid vaccinecomprises more than one immunogenic epitope. In some embodiments, thenucleic acid vaccine is a DNA-based vaccine. In some embodiments, thenucleic acid vaccine is a RNA-based vaccine. In some embodiments, theRNA-based vaccine comprises mRNA. In some embodiments, the RNA-basedvaccine comprises naked mRNA. In some embodiments, the RNA-based vaccinecomprises modified mRNA (e.g., mRNA protected from degradation usingprotamine. mRNA containing modified 5′ CAP structure or mRNA containingmodified nucleotides). In some embodiments, the RNA-based vaccinecomprises single-stranded mRNA.

The polynucleotide may be substantially pure, or contained in a suitablevector or delivery system. Suitable vectors and delivery systems includeviral, such as systems based on adenovirus, vaccinia virus,retroviruses, herpes virus, adeno-associated virus or hybrids containingelements of more than one virus. Non-viral delivery systems includecationic lipids and cationic polymers (e.g., cationic liposomes).

One or more neoantigen peptides can be encoded and expressed in vivousing a viral based system. Viral vectors may be used as recombinantvectors in the present disclosure, wherein a portion of the viral genomeis deleted to introduce new genes without destroying infectivity of thevirus. The viral vector of the present disclosure is a nonpathogenicvirus. In some embodiments the viral vector has a tropism for a specificcell type in the mammal. In another embodiment, the viral vector of thepresent disclosure is able to infect professional antigen presentingcells such as dendritic cells and macrophages. In yet another embodimentof the present disclosure, the viral vector is able to infect any cellin the mammal. The viral vector may also infect tumor cells. Viralvectors used in the present disclosure include but is not limited toPoxvirus such as vaccinia virus, avipox virus, fowlpox virus and ahighly attenuated vaccinia virus (Ankara or MVA), retrovirus,adenovirus, baculovirus and the like.

A vaccine can be delivered via a variety of routes. Delivery routes caninclude oral (including buccal and sub-lingual), rectal, nasal, topical,transdermal patch, pulmonary, vaginal, suppository, or parenteral(including intramuscular, intra-arterial, intrathecal, intradermal,intraperitoneal, subcutaneous and intravenous) administration or in aform suitable for administration by aerosolization, inhalation orinsufflation. General information on drug delivery systems can be foundin Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems(Lippencott Williams & Wilkins, Baltimore Md. (1999). The vaccinedescribed herein can be administered to muscle, or can be administeredvia intradermal or subcutaneous injections, or transdermally, such as byiontophoresis. Epidermal administration of the vaccine can be employed.

In some instances, the vaccine can also be formulated for administrationvia the nasal passages. Formulations suitable for nasal administration,wherein the carrier is a solid, can include a coarse powder having aparticle size, for example, in the range of about 10 to about 500microns which is administered in the manner in which snuff is taken,i.e., by rapid inhalation through the nasal passage from a container ofthe powder held close up to the nose. The formulation can be a nasalspray, nasal drops, or by aerosol administration by nebulizer. Theformulation can include aqueous or oily solutions of the vaccine.

The vaccine can be a liquid preparation such as a suspension, syrup orelixir. The vaccine can also be a preparation for parenteral,subcutaneous, intradermal, intramuscular or intravenous administration(e.g., injectable administration), such as a sterile suspension oremulsion.

The vaccine can include material for a single immunization, or mayinclude material for multiple immunizations (i.e. a ‘multidose’ kit).The inclusion of a preservative is preferred in multidose arrangements.As an alternative (or in addition) to including a preservative inmultidose compositions, the compositions can be contained in a containerhaving an aseptic adaptor for removal of material.

The vaccine can be administered in a dosage volume of about 0.5 mL,although a half dose (i.e. about 0.25 mL) can be administered tochildren. Sometimes the vaccine can be administered in a higher dosee.g. about 1 ml.

The vaccine can be administered as a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore dose-course regimen. Sometimes, the vaccine is administered as a 1,2, 3, or 4 dose-course regimen. Sometimes the vaccine is administered asa 1 dose-course regimen. Sometimes the vaccine is administered as a 2dose-course regimen.

The administration of the first dose and second dose can be separated byabout 0 day, 1 day, 2 days, 5 days, 7 days, 14 days, 21 days, 30 days, 2months, 4 months, 6 months, 9 months, 1 year, 1.5 years, 2 years, 3years, 4 years, or more.

The vaccine described herein can be administered every 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more years. Sometimes, the vaccine described herein isadministered every 2, 3, 4, 5, 6, 7, or more years. Sometimes, thevaccine described herein is administered every 4, 5, 6, 7, or moreyears. Sometimes, the vaccine described herein is administered once.

The dosage examples are not limiting and are only used to exemplifyparticular dosing regiments for administering a vaccine describedherein. The effective amount for use in humans can be determined fromanimal models. For example, a dose for humans can be formulated toachieve circulating, liver, topical and/or gastrointestinalconcentrations that have been found to be effective in animals. Based onanimal data, and other types of similar data, those skilled in the artcan determine the effective amounts of a vaccine composition appropriatefor humans.

The effective amount when referring to an agent or combination of agentswill generally mean the dose ranges, modes of administration,formulations, etc., that have been recommended or approved by any of thevarious regulatory or advisory organizations in the medical orpharmaceutical arts (e.g., FDA, AMA) or by the manufacturer or supplier.

In some aspects, the vaccine and kit described herein can be stored atbetween 2° C. and 8° C. In some instances, the vaccine is not storedfrozen. In some instances, the vaccine is stored in temperatures of suchas at −20° C. or −80° C. In some instances, the vaccine is stored awayfrom sunlight.

IX. Kits

The neoantigen therapeutic described herein can be provided in kit formtogether with instructions for administration. Typically the kit wouldinclude the desired neoantigen therapeutic in a container, in unitdosage form and instructions for administration. Additionaltherapeutics, for example, cytokines, lymphokines, checkpointinhibitors, antibodies, can also be included in the kit. Other kitcomponents that can also be desirable include, for example, a sterilesyringe, booster dosages, and other desired excipients.

Kits and articles of manufacture are also provided herein for use withone or more methods described herein. The kits can contain one or moreneoantigenic polypeptides comprising one or more neoepitopes. The kitscan also contain nucleic acids that encode one or more of the peptidesor proteins described herein, antibodies that recognize one or more ofthe peptides described herein, or APC-based cells activated with one ormore of the peptides described herein. The kits can further containadjuvants, reagents, and buffers necessary for the makeup and deliveryof the vaccines.

The kits can also include a carrier, package, or container that iscompartmentalized to receive one or more containers such as vials,tubes, and the like, each of the container(s) comprising one of theseparate elements, such as the peptides and adjuvants, to be used in amethod described herein. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers can be formedfrom a variety of materials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, bags, containers, bottles,and any packaging material suitable for a selected formulation andintended mode of administration and treatment. A kit typically includeslabels listing contents and/or instructions for use, and package insertswith instructions for use. A set of instructions will also typically beincluded.

The present disclosure will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes, and are not intended to limit the present disclosure in anymanner. Those of skill in the art will readily recognize a variety ofnon-critical parameters that can be changed or modified to yieldalternative embodiments according to the present disclosure. Allpatents, patent applications, and printed publications listed herein areincorporated herein by reference in their entirety.

Examples

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1—Induction of CD4⁺ and CD8⁺ T Cell Responses

In vitro T cell inductions are used to expand neo-antigen specific Tcells. Mature professional APCs are prepared for these assays in thefollowing way. Monocytes are enriched from healthy human donor PBMCsusing a bead-based kit (Miltenyi). Enriched cells are plated in GM-CSFand IL-4 to induce immature DCs. Enriched cells may also be plated usingFLT-3L and IL-4 to induce immature DCs. After 5 days, immature DCs areincubated at 37° C. with pools of peptides for 1 hour before addition ofa cytokine maturation cocktail (GM-CSF, IL-1β, IL-4, IL-6, TNFα, PGE1β).The maturation cocktail may in some cases include FLT-3L, IL-1β, IL-4,IL-6, TNFα, PGE1β. The pools of peptides can include multiple mutations,with both shortmers and longmers to expand CD8⁺ and CD4⁺ T cells,respectively. Long peptides were also used to demonstrate thepossibility to stimulate CD8+ cells in this setting, also shown in FIG.3C. Cells are incubated at 37° C. to mature DCs.

After maturation of DCs, PBMCs (either bulk or enriched for T cells) areadded to mature dendritic cells with proliferation cytokines. Culturesare monitored for peptide-specific T cells using a combination offunctional assays and/or tetramer staining. Parallel immunogenicityassays with the modified and parent peptides allowed for comparisons ofthe relative efficiency with which the peptides expandedpeptide-specific T cells.

Example 2—Tetramer Staining Assay

MHC tetramers are purchased or manufactured on-site, and are used tomeasure peptide-specific T cell expansion in the immunogenicity assays.For the assessment, tetramer is added to 1×10⁵ cells in PBS containing1% FCS and 0.1% sodium azide (FACS buffer) according to manufacturer'sinstructions. Cells are incubated in the dark for 20 minutes at roomtemperature. Antibodies specific for T cell markers, such as CD8, arethen added to a final concentration suggested by the manufacturer, andthe cells are incubated in the dark at 4° C. for 20 minutes. Cells arewashed with cold FACS buffer and resuspended in buffer containing 1%formaldehyde. Cells are acquired on a FACS Calibur (Becton Dickinson)instrument, and are analyzed by use of Cellquest software (BectonDickinson). For analysis of tetramer positive cells, the lymphocyte gateis taken from the forward and side-scatter plots. Data are reported asthe percentage of cells that were CD8⁺/tetramer⁺.

Example 3—Intracellular Cytokine Staining Assay

In the absence of well-established tetramer staining to identifyantigen-specific T cell populations, antigen-specificity can beestimated using assessment of cytokine production using well-establishedflow cytometry assays. Briefly, T cells are stimulated with the peptideof interest and compared to a control. After stimulation, production ofcytokines by CD4⁺ T cells (e.g., IFNγ and TNFα) are assessed byintracellular staining. These cytokines, especially IFNγ, can be used toidentify stimulated cells. FACS analysis of antigen-specific inductionof IFNγ and TNFα levels of CD4+ cells from a healthy donor stimulatedwith APCs loaded with or without a mutant RAS peptide was performed.

Example 4—ELISPOT Assay

Peptide-specific T cells are functionally enumerated using the ELISPOTassay (BD Biosciences), which measures the release of IFNγ from T cellson a single cell basis. Target cells (T2 or HLA-A0201 transfected C1Rs)were pulsed with 10 μM peptide for 1 hour at 37° C., and washed threetimes. 1×10⁵ peptide-pulsed targets are co-cultured in the ELISPOT platewells with varying concentrations of T cells (5×10² to 2×10³) taken fromthe immunogenicity culture. Plates are developed according to themanufacturer's protocol, and analyzed on an ELISPOT reader (CellularTechnology Ltd.) with accompanying software. Spots corresponding to thenumber of IFNγ-producing T cells are reported as the absolute number ofspots per number of T cells plated. T cells expanded on modifiedpeptides are tested not only for their ability to recognize targetspulsed with the modified peptide, but also for their ability torecognize targets pulsed with the parent peptide. The IFNγ levels ofsamples mock transduced or transduced with a lentiviral expressionvector encoding a mutant RAS peptide were determined.

Example 5—CD107 Staining Assay

CD107a and b are expressed on the cell surface of CD8⁺ T cells followingactivation with cognate peptide. The lytic granules of T cells have alipid bilayer that contains lysosomal-associated membrane glycoproteins(“LAMPs”), which include the molecules CD107a and b. When cytotoxic Tcells are activated through the T cell receptor, the membranes of theselytic granules mobilize and fuse with the plasma membrane of the T cell.The granule contents are released, and this leads to the death of thetarget cell. As the granule membrane fuses with the plasma membrane,C107a and b are exposed on the cell surface, and therefore are markersof degranulation. Because degranulation as measured by CD107a and bstaining is reported on a single cell basis, the assay is used tofunctionally enumerate peptide-specific T cells. To perform the assay,peptide is added to HLA-A02:01-transfected cells C1R to a finalconcentration of 20 μM, the cells were incubated for 1 hour at 37° C.,and washed three times. 1×10⁵ of the peptide-pulsed C1R cells werealiquoted into tubes, and antibodies specific for CD107a and b are addedto a final concentration suggested by the manufacturer (BectonDickinson). Antibodies are added prior to the addition of T cells inorder to “capture” the CD107 molecules as they transiently appear on thesurface during the course of the assay. 1×10⁵ T cells from theimmunogenicity culture are added next, and the samples were incubatedfor 4 hours at 37° C. The T cells are further stained for additionalcell surface molecules such as CD8 and acquired on a FACS Caliburinstrument (Becton Dickinson). Data is analyzed using the accompanyingCellquest software, and results are reported as the percentage ofCD8⁺/CD107a and b⁺ cells.

Example 6—Cytotoxicity Assays

Cytotoxic activity is measured using a chromium release assay. Target T2cells are labeled for 1 hour at 37° C. with Na⁵¹Cr and washed 5×10³target T2 cells were then added to varying numbers of T cells from theimmunogenicity culture. Chromium release is measured in supernatantharvested after 4 hours of incubation at 37° C. The percentage ofspecific lysis is calculated as: Experimental release-spontaneousrelease/Total release-spontaneous release×100.

Cytotoxicity activity is measured with the detection of cleaved Caspase3 in target cells by Flow cytometry. Target cancer cells are engineeredto express the mutant peptide along with the proper MHC-I allele.Mock-transduced target cells (i.e. not expressing the mutant peptide)are used as a negative control. The cells are labeled with CFSE todistinguish them from the stimulated PBMCs used as effector cells. Thetarget and effector cells are co-cultured for 6 hours before beingharvested. Intracellular staining is performed to detect the cleavedform of Caspase 3 in the CFSE-positive target cancer cells. Thepercentage of specific lysis is calculated as: Experimental cleavage ofCaspase 3/spontaneous cleavage of Caspase 3 (measured in the absence ofmutant peptide expression)×100.

In some examples, cytotoxicity activity is assessed by co-culturing Tcells expressing a TCR specific to a mutant RAS peptide on a specificHLA, with mutant RAS peptide-transduced target cancer cells expressingthe corresponding HLA, and by determining the relative growth of thetarget cells, along with measuring the apoptotic marker Annexin V in thetarget cancer cells specifically. Target cancer cells are engineered toexpress the mutant peptide along with the proper MHC-I allele.Mock-transduced target cells (i.e. not expressing the mutant peptide)are used as a negative control. The cells are also transduced to stablyexpress GFP allowing the tracking of target cell growth. PBMCs fromhealthy donors, used as effector cells, are transduced to express a TCRspecific to a mutant RAS peptide. Mock-transduced cells are used as anegative control. The target cells are cocultured with different amountof effector cells for 72 h in media containing Annexin V-detectionreagent. The GFP signal and Annexin-V signal are measured over time withan IncuCyte S3 apparatus. Annexin V signal originating from effectorcells is filtered out by size exclusion. Target cell growth and death isexpressed as GFP and Annexin-V area (mm²) over time, respectively.

Example 7—Enhanced CD8⁺ T Cell Responses In Vivo Using Longmers andShortmers Sequentially

Vaccination with longmer peptides can induce both CD4⁺ and CD8⁺ T cellresponses, depending on the processing and presentation of the peptides.Vaccination with minimal shortmer epitopes focuses on generating CD8⁺ Tcell responses, but does not require peptide processing before antigenpresentation. As such, any cell can present the epitope readily, notjust professional antigen-presenting cells (APCs). This may lead totolerance of T cells that come in contact with healthy cells presentingantigens as part of peripheral tolerance. To circumvent this, initialimmunization with longmers allows priming of CD8⁺ T cells only by APCsthat can process and present the peptides. Subsequent immunizationsboosts the initial CD8⁺ T cell responses.

In Vivo Immunogenicity Assays

Nineteen 8-12 week old female C57BL/6 mice (Taconic Biosciences) wererandomly and prospectively assigned to treatment groups on arrival.Animals were acclimated for three (3) days prior to study commencement.Animals were maintained on LabDiet™ 5053 sterile rodent chow and sterilewater provided ad libitum. Animals in Group 1 served as vaccinationadjuvant-only controls and were administered polyinosinic:polycytidylicacid (polyI:C) alone at 100 μg in a volume of 0.1 mL administered viasubcutaneous injection (s.c.) on day 0, 7, and 14. Animals in Group 2were administered 50 μg each of six longmer peptides (described below)along with polyI:C at 100 μg s.c. in a volume of 0.1 mL on day 0, 7 and14. Animals in Group 3 were administered 50 μg each of six longmerpeptides (described below) along with polyI:C at 100 μg s.c. in a volumeof 0.1 mL on day 0 and molar-matched equivalents of correspondingshortmer peptides (described below) along with polyI:C at 100 μg s.c. ina volume of 0.1 mL on day 7 and 14. Animals were weighed and monitoredfor general health daily. Animals were euthanized by CO2 overdose atstudy completion Day 21, if an animal lost >30% of its body weightcompared to weight at Day 0; or if an animal was found moribund. Atsacrifice, spleens were harvested and processed into single-cellsuspensions using standard protocols. Briefly, spleens were mechanicaldegraded through a 70 μM filter, pelleted, and lysed with ACK lysisbuffer (Sigma) before resuspension in cell culture media.

Peptides

Six previously identified murine neoantigens were used based on theirdemonstrated ability to induce CD8⁺ T cell responses. For eachneoantigen, shortmers (8-11 amino acids) corresponding to the minimalepitope have been defined. Longmers corresponding to 20-27 amino acidssurrounding the mutation were used.

ELISPOT

ELISPOT analysis (Mouse IFNγ ELISPOT Reasy-SET-Go; EBioscience) wasperformed according to the kit protocol. Briefly, one day prior to dayof analysis, 96-well filter plates (0.45 μm pore size hydrophobic PVDFmembrane; EMD Millipore) were activated (35% EtOH), washed (PBS) andcoated with capture antibody (1:250; 4° C. O/N). On the day of analysis,wells were washed and blocked (media; 2 hours at 37° C.). Approximately2×10⁵ cells in 100 μL was added to the wells along with 100 μL of 10 mMtest peptide pool (shortmers), or PMA/ionomycin positive controlantigen, or vehicle. Cells incubated with antigen overnight (16-18hours) at 37° C. The next day, the cell suspension was discarded, andwells were washed once with PBS, and twice with deionized water. For allwash steps in the remainder of the assay, wells were allowed to soak for3 minutes at each wash step. Wells were then washed three times withwash buffer (PBS+0.05% Tween-20), and detection antibody (1:250) wasadded to all wells. Plates were incubated for two hours at roomtemperature. The detection antibody solution was discarded, and wellswere washed three times with wash buffer. Avidin-HRP (1:250) was addedto all wells, and plates were incubated for one hour at roomtemperature. Conjugate solution was discarded, and wells washed threetimes with wash buffer, then once with PBS. Substrate(3-amino-9-ethyl-carbazole, 0.1 M Acetate buffer, H₂O₂) was added to allwells, and spot development monitored (approximately 10 minutes).Substrate reaction was stopped by washing wells with water, and plateswere allowed to air-dry overnight. The plates were analyzed on anELISPOT reader (Cellular Technology Ltd.) with accompanying software.Spots corresponding to the number of IFNγ-producing T cells are reportedas the absolute number of spots per number of T cells plated.

Example 8—Detection of Mutant RAS Peptides by Mass Spectrometry

293T cells were transduced with a lentiviral vector encoding variousregions of a mutant RAS peptide. 43 million of the transduced cellsexpressing peptides encoded by the a mutant RAS peptide were culturedand peptides were eluted from HLA-peptide complexes using an acid wash.Eluted peptides were then analyzed by MS/MS. For 293T cells expressingan HLA-A11:01 protein, the peptide VVVGACGVGK, was detected by massspectrometry (FIG. 2). For 293T cells expressing an HLA-A11:01 protein,the peptides VVVGAVGVGK and VVGAVGVGK, was detected by mass spectrometry(FIG. 2).

Example 9—Mutant KRAS Peptides Produce Strong Epitopes on MultipleAlleles

Multiple peptides containing the neoepitopes in the table below wereexpressed or loaded onto antigen presenting cells (APCs). Massspectrometry was then performed and the affinity of the neoepitopes forthe indicated HLA alleles and stability of the neoepitopes with the HLAalleles was determined.

Example 10—Multiple Neoepitopes Elicit CD8+ T Cell Responses

PBMC samples from a human donor were used to perform antigen specific Tcell induction. CD8⁺ T cell inductions were analyzed after manufacturingT cells. Cell samples can be taken out at different time points foranalysis. pMHC multimers were used to monitor the fraction of antigenspecific CD8⁺ T cells in the induction cultures. FIGS. 3A-C depictexemplary results showing the fraction of antigen specific CD8⁺ T cellsinduced with a RAS G12V, a RAS G12 C and a RAS G12D mutant peptide. Longpeptides were also used to demonstrate the possibility to stimulate CD8+cells, as shown in FIG. 3C.

Measured Measured Peptide Peptide Affinity stability Gene HLA AlleleSequence Length (nM) (hr) KRAS, G12C A02.01 LVVVGACGV 9 667.1 0.6KRAS, G12C A02.01 KLVVVGACGV 10 70.3 1.0 KRAS, G12D A02.01 LVVVGADGV 9977.4 0.0 KRAS, G12D A02.01 KLVVVGADGV 10 137.7 0.9 KRAS, G12V A02.01LVVVGAVGV 9 682.5 0.6 KRAS, G12V A02.01 KLVVVGAVGV 10 57.6 0.9KRAS, G12C A03.01 VVGACGVGK 9 4.1 5.0 KRAS, G12C A03.01 VVVGACGVGK 101.6 2.5 KRAS, G12D A03.01 VVGADGVGK 9 518.7 NB KRAS, G12D A03.01VVVGADGVGK 10 314.9 2.3 KRAS, G12V A03.01 VVGAVGVGK 9 1.9 1.2 KRAS, G12VA03.01 VVVGAVGVGK 10 44.2 6.7 KRAS, G12C A11.01 VVGACGVGK 9 43.2 10.0KRAS, G12C A11.01 VVVGACGVGK 10 69.3 15.7 KRAS, G12D A11.01 VVGADGVGK 9203.9 3.4 KRAS, G12D A11.01 VVVGADGVGK 10 33.1 13.0 KRAS, G12V A11.01VVGAVGVGK 9 7.7 16.9 KRAS, G12V A11.01 VVVGAVGVGK 10 26.1 24.3

Example 11—Cytotoxicity Assay of Induced T Cells

A cytotoxicity assay was used to assess whether the induced T cellcultures can kill antigen expressing tumor lines. In this example,expression of active caspase 3 on alive and dead tumor cells wasmeasured to quantify early cell death and dead tumor cells. In FIG. 4B,the induced CD8⁺ responses were capable of killing antigen expressingtumor targets. The percent live caspase-A positive target cells ofsamples mock transduced or transduced with a lentiviral expressionvector encoding a mutant RAS peptide is shown.

Example 12—Mutant KRAS Peptide Stablemers Produce Strong Epitopes onMultiple Alleles

Multiple peptides containing the neoepitopes in the table below wereexpressed or loaded onto antigen presenting cells (APCs). Massspectrometry was then performed and the affinity of the neoepitopes forthe indicated HLA alleles and stability of the neoepitopes with the HLAalleles was determined.

Predicted Measured Measured HLA Peptide affinity Affinity stability GeneClass Allele Sequence nM (nM) (hr) KRAS, G12C Neoepitope A02.01KLVVVGACGV 203.6 70.3 1.0 KRAS, G12C Affinity A02.01 FLVVVGACGL 181.869.4 1.4 Enh. KRAS, G12C Affinity A02.01 FMVVVGACGI 173.8 17.0 1.0 Enh.KRAS, G12C Affinity A02.01 FLVVVGACGI 194.9 27.8 1.4 Enh. KRAS, G12CAffinity A02.01 FMVVVGACGV 53.2 1.1 5.4 Enh. KRAS, G12C Affinity A02.01FLVVVGACGV 61.6 4.2 7.4 Enh. KRAS, G12C Affinity A02.01 MLVVVGACGV 117.718.2 1.7 Enh. KRAS, G12C Affinity A02.01 FMVVVGACGL 167.3 85.3 0.9 Enh.KRAS, G12C Affinity A02.01 YLVVVGACGV 80.7 3.9 9.3 Enh. KRAS, G12CAffinity A02.01 KMVVVGACGV 189.3 11.9 3.8 Enh. KRAS, G12C AffinityA02.01 YMVVVGACGV 70.1 7.0 6.4 Enh. KRAS, G12C Affinity A02.01MMVVVGACGV 95.8 18.9 2.1 Enh.

What is claimed is:
 1. A composition comprising: (a) at least onepolypeptide or a pharmaceutically acceptable salt thereof comprising twoor more mutant RAS peptide sequences selected from the group consistingof; (i) KLVVVGADGV, KLVVVGACGV, KLVVVGAVGV, LVVVGADGV, LVVVGACGV,LVVVGAVGV; (ii) GADGVGKSAL, GACGVGKSAL, GAVGVGKSAL, GADGVGKSA,GACGVGKSA, GAVGVGKSA; and/or (iii) VVGADGVGK, VVGACGVGK, VVGAVGVGK,VVVGADGVGK, VVVGACGVGK, VVVGAVGVGK; or (b) at least one polynucleotideencoding the at least one polypeptide.
 2. The composition of claim 1,wherein the composition comprises a mixture of the three or more mutantRAS peptide sequences.
 3. A composition comprising: (a) at least onepolypeptide or a pharmaceutically acceptable salt thereof comprising twoor more mutant RAS peptide sequences each comprising: (i) at least 8contiguous amino acids of a mutant RAS protein comprising a mutation atG12, and (ii) the mutation at G12; and further wherein three or moreamino acid residues that are heterologous to the mutant RAS protein arelinked to the N-terminus or C-terminus of the two or more mutant RASpeptide sequences, wherein the three or more amino acid residues enhanceprocessing of the mutant RAS peptide sequences in cell and/or enhancepresentation of an epitope of the mutant RAS peptide sequences; or (b)at least one polynucleotide encoding the at least one polypeptide. 4.The composition of claim 3, wherein the three or more amino acidresidues that are heterologous to the mutant RAS protein are linked tothe N-terminus or C-terminus of the two or more mutant RAS peptidesequences comprises an amino acid sequence of a protein of CMV such aspp65, HIV, or MART-1.
 5. The composition of claim 3 or 4, wherein thethree or more amino acid residues that are heterologous to the mutantRAS protein are linked to the N-terminus or C-terminus of the two ormore mutant RAS peptide sequences comprises at least 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.6. The composition of any one of claims 3-5, wherein the three or moreamino acid residues that are heterologous to the mutant RAS protein arelinked to the N-terminus or C-terminus of the two or more mutant RASpeptide sequences comprises at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, or 100 amino acids.7. A composition comprising (a) at least one polypeptide or apharmaceutically acceptable salt thereof of the formula(Xaa_(N))_(N)-(Xaa_(RAS))_(P)-(Xaa_(C))_(C) wherein P is an integergreater than 7; (Xaa_(RAS))_(P) is a mutant RAS peptide sequencecomprising at least 8 contiguous amino acids of a mutant RAS protein;the at least 8 contiguous amino acids comprising at least 8 contiguousamino acids of the sequenceLys₁ Leu₂ Val₃ Val₄ Val₅ Gly₆ Ala₇ Xaa₈ Gly₉ Val₁₀ Gly₁₁ Lys₁₂ Ser₁₃Ala₁₄Leu₁₅ N is (i) 0 or (ii) an integer greater than 2; (Xaa_(N))_(N)is any amino acid sequence heterologous to the mutant RAS protein; C is(i) 0 or (ii) an integer greater than 2; (Xaa_(C))_(C) is any amino acidsequence heterologous to the mutant RAS protein; Xaa₈ is selected fromthe group consisting of Asp, Val, Cys, Ala, Arg and Ser; the polypeptideis not KLVVVGAVGVGKSALTIQL; and wherein if N is 0 C is not 0 and if C is0 N is not 0; or (b) at least one polynucleotide encoding the at leastone polypeptide.
 8. The composition of claim 7, wherein (Xaa)_(N) and/or(Xaa_(C))_(C) comprises an amino acid sequence of a protein of CMV suchas pp65, HIV, or MART-1.
 9. The composition of claim 7 or 8, wherein Nand/or C is an integer greater than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, or
 40. 10. The composition of any one ofclaims 7-9, wherein N and/or C is an integer less than 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90,or
 100. 11. The composition of any one of claims 7-10, wherein N is O.12. The composition of any one of claims 7-10, wherein C is
 0. 13. Acomposition comprising (a) at least one polypeptide or apharmaceutically acceptable salt thereof comprising of an amino acidsequence of Xaa₁-Xaa₂-Val₃-Val₄-Val₅-Gly₆-Ala₇-Xaa₈-Gly₉-Xaa₁₀

wherein Xaa₁ is not Ala; with the proviso that when Xaa₁ is not Lys,Xaa₂ is Leu and/or Xaa₁₀ is Gly; Xaa₂ is not Glu; with the proviso thatwhen Xaa₂ is not Leu, Xaa₁ is Lys and/or Xaa₁₀ is Gly; Xaa₈ is selectedfrom the group consisting of Asp, Val, Cys, Ala, Arg and Ser; with theproviso that when Xaa₈ is Glu, Xaa₁ is not Tyr and/or Xaa₂ is not Leu,and with the proviso that when Xaa₈ is Val, Xaa₁ is not Lys; Xaa₁₀ isany amino acid; with the proviso that when Xaa₁₀ is not Gly, Xaa₁ is Lysand/or Xaa₂ is Leu; and the polypeptide comprises anHLA-A02:01-restricted T cell epitope, HLA-A03:01-restricted T cellepitope, an HLA-A11:01-restricted T cell epitope, anHLA-A03:02-restricted T cell epitope, an HLA-A30:01-restricted T cellepitope, an HLA-A31:01-restricted T cell epitope, anHLA-A33:01-restricted T cell epitope, an HLA-A33:03-restricted T cellepitope, an HLA-A68:01-restricted T cell epitope, or anHLA-A74:01-restricted T cell epitope that binds to an HLA-A02:01,HLA-A03:01, HLA-A11:01, HLA-A03:02, HLA-A30:01, HLA-A31:01, HLA-A33:01,HLA-A33:03, HLA-A68:01, and/or an HLA-A74:01 molecule; and induces anHLA-A02:01-restricted cytotoxic T cell response, anHLA-A02:01-restricted cytotoxic T cell response, HLA-A03:01-restrictedcytotoxic T cell response, an HLA-A11:01-restricted cytotoxic T cellresponse, an HLA-A03:02-restricted cytotoxic T cell response, anHLA-A30:01-restricted cytotoxic T cell response, anHLA-A31:01-restricted cytotoxic T cell response, anHLA-A33:01-restricted cytotoxic T cell response, anHLA-A33:03-restricted cytotoxic T cell response, anHLA-A68:01-restricted cytotoxic T cell response, or anHLA-A74:01-restricted cytotoxic T cell response; and that binds to anHLA-A02:01, HLA-A03:01, HLA-A11:01, HLA-A03:02, HLA-A30:01, HLA-A31:01,HLA-A33:01, HLA-A33:03, HLA-A68:01, and/or an HLA-A74:01; or (b) atleast one polynucleotide encoding the at least one polypeptide.
 14. Acomposition comprising: (a) at least one polypeptide or apharmaceutically acceptable salt thereof comprising one or more mutantRAS peptide sequences each comprising: (i) at least 8 contiguous aminoacids of a mutant RAS protein comprising a G12A, G12C, G12D, G12R, G12S,or G12V mutation, and (ii) the G12A, G12C, G12D, G12R, G12S, or G12Vmutation; and further wherein the peptide: (i) comprises a mutation notencoded by a genome of a cancer cell and has an affinity or predictedaffinity of 150 nM or less for an HLA-A02:01 allele and/or a half-lifeof 2 hours or more, or (j) has a half life of 2 hours or more and anaffinity or predicted affinity of 150 nM or less for an HLA-A02:01allele, an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01allele, an HLA-A33:03 allele, an HLA-A68:01 allele, or an HLA-A74:01allele and/or an HLA-008:02 allele; or (b) at least one polynucleotideencoding the at least one polypeptide.
 15. The composition of any one ofclaims 1 to 14, wherein the composition further comprises (i) a peptidecomprising a peptide sequence in any one of Table 3 to 14, or (ii) apolynucleotide encoding the peptide comprising a sequence in Table 3 to14.
 16. A composition comprising: (a) at least one polypeptide or apharmaceutically acceptable salt thereof comprising one or more mutantRAS peptide sequences selected from the group consisting of: DTAGHEEY,TAGHEEYSAM, DILDTAGHE, DILDTAGH, ILDTAGHEE, ILDTAGHE, DILDTAGHEEY,DTAGHEEYS, LLDILDTAGH, DILDTAGRE, DILDTAGR, ILDTAGREE, ILDTAGRE,CLLDILDTAGR, TAGREEYSAM, REEYSAMRD, DTAGKEEYSAM, CLLDILDTAGK, DTAGKEEY,LLDILDTAGK, ILDTAGKE, ILDTAGKEE, DTAGLEEY, ILDTAGLE, DILDTAGL,ILDTAGLEE, GLEEYSAMRDQY, LLDILDTAGLE, LDILDTAGL, DILDTAGLE, DILDTAGLEEY,AGVGKSAL, GAAGVGKSAL, AAGVGKSAL, CGVGKSAL, ACGVGKSAL, DGVGKSAL,ADGVGKSAL, DGVGKSALTI, GARGVGKSA, KLVVVGARGV, VVVGARGV, SGVGKSAL,VVVGASGVGK, GASGVGKSAL, VGVGKSAL, VVVGAGCVGK, KLVVVGAGC, GDVGKSAL,DVGKSALTI, VVVGAGDVGK, TAGKEEYSAM, DTAGHEEYSAM, TAGHEEYSA, DTAGREEYSAM,TAGKEEYSA, AAGVGKSA, AGCVGKSAL, AGDVGKSAL, AGKEEYSAMR, AGVGKSALTI,ARGVGKSAL, ASGVGKSA, ASGVGKSAL, AVGVGKSA, CVGKSALTI, DILDTAGK,DILDTAGREEY, DTAGHEEYSAMR, DTAGKEEYS, DTAGKEEYSAMR, DTAGLEEYS,DTAGLEEYSA, DTAGLEEYSAMR, DTAGREEYS, DTAGREEYSAMR, GAAGVGKSA, GACGVGKSA,GACGVGKSAL, GADGVGKS, GAGDVGKSA, GAGDVGKSAL, GASGVGKSA, GCVGKSAL,GCVGKSALTI, GHEEYSAM, GKEEYSAM, GLEEYSAMR, GREEYSAM, GREEYSAMR,HEEYSAMRD, KEEYSAMRD, KLVVVGASG, LDILDTAGR, LEEYSAMRD, LVVVGARGV,LVVVGASGV, REEYSAMRDQY, RGVGKSAL, TAGLEEYSA, TEYKLVVVGAA, VGAAGVGKSA,VGADGVGK, VGASGVGKSA, VGVGKSALTI, VVVGAAGV, VVVGAVGV, YKLVVVGAC,YKLVVVGAD, YKLVVVGAR, and DILDTAGKE; or (b) at least one polynucleotideencoding the at least one polypeptide.
 17. The composition of claim 16,wherein the composition further comprises; (i) a peptide comprising apeptide sequence in any one of Tables 1 to 14, or (ii) a polynucleotideencoding the peptide comprising a sequence in Tables 1 to
 14. 18. Thecomposition of any one of claims 1-17, wherein at least one of themutant RAS peptide sequences comprises N or C terminal amino acidsequence extension of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, or 40 amino acids, wherein the N or Cterminal extension is a wild-type RAS amino acid sequence or anon-heterologous RAS amino acid sequence.
 19. The composition of any oneof claims 1-18, wherein the at least one polypeptide comprises at least3, 4, 5, 6, 7, 8, 9, or 10 mutant RAS peptide sequences.
 20. Thecomposition of any one of claims 1-19, wherein the at least onepolypeptide comprises at least two polypeptides, or the at least onepolynucleotide comprises at least two polynucleotides.
 21. Thecomposition of any one of claims 1-20, wherein at least one of themutant RAS peptide sequences comprises at least 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, or 40 contiguous amino acids of a mutant RASprotein.
 22. The composition of any one of claims 1-21, wherein at least2, 3, 4, 5, 6, 7, 8, 9, or 10 of the mutant RAS peptide sequencescomprise at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or40 contiguous amino acids of a mutant RAS protein.
 23. The compositionof any one of claims 1-22, wherein each of the mutant RAS peptidesequences or each of the two or more RAS peptide sequences comprises atleast 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguousamino acids of a mutant RAS protein.
 24. The composition of any one ofclaims 1-23, wherein the at least one polypeptide comprises at least onemutant RAS peptide sequence that binds to or is predicted to bind to aprotein encoded by an HLA-A02:01 allele, an HLA-A03:01 allele, anHLA-A11:01 allele, and/or an HLA-008:02 allele.
 25. The composition ofany one of claims 1-24, wherein the at least one polypeptide comprisesat least one mutant RAS peptide sequence that binds to or is predictedto bind to a protein encoded by: (a) an HLA-A02:01 allele and anHLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, anHLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, anHLA-A33:03 allele, an HLA-A68:01 allele, or an HLA-A74:01 allele; (b) anHLA-A02:01 allele and an HLA-008:02 allele; (c) an HLA-A03:01 allele, anHLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, anHLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, anHLA-A68:01 allele, or an HLA-A74:01 allele and an HLA-008:02 allele; or(d) an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, anHLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, anHLA-A33:03 allele, an HLA-A68:01 allele, or an HLA-A74:01 allele andallele and an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01allele, an HLA-A33:03 allele, an HLA-A68:01 allele, or an HLA-A74:01allele.
 26. The composition of any one of claims 1-25, wherein themutant RAS peptide sequences comprise; (a) a first mutant RAS peptidesequence that binds to or is predicted to bind to a protein encoded byan HLA-A02:01 allele, an HLA-A03:01 allele, an HLA-A11:01 allele, anHLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, anHLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, anHLA-A74:01 allele, and/or an HLA-008:02 allele; and (b) a second RASpeptide sequence that binds to or is predicted to bind to a proteinencoded by an HLA-A02:01 allele, an HLA-A03:01 allele, an HLA-A11:01allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01allele, an HLA-A74:01 allele, and/or an HLA-008:02 allele; wherein, thefirst mutant RAS peptide sequence binds to or is predicted to bind to aprotein encoded by different HLA allele than the second mutant RASpeptide sequence.
 27. The composition of any one of claims 1-26, whereinthe at least one polypeptide comprises at least one mutant RAS peptidesequence that binds to a protein encoded by an HLA allele with anaffinity of less than 10 μM, less than 1 μM, less than 500 nM, less than400 nM, less than 300 nM, less than 250 nM, less than 200 nM, less than150 nM, less than 100 nM, or less than 50 nM.
 28. The composition of anyone of claims 1-27, wherein the at least one polypeptide comprises atleast one mutant RAS peptide sequence that binds to a protein encoded byan HLA allele with a stability of greater than 24 hours, greater than 12hours, greater than 9 hours, greater than 6 hours, greater than 5 hours,greater than 4 hours, greater than 3 hours, greater than 2 hours,greater than 1 hour, greater than 45 minutes, greater than 30 minutes,greater than 15 minutes, or greater than 10 minutes.
 29. The compositionof claim 27 or 28, wherein the HLA allele is selected from the groupconsisting of HLA-A02:01 allele, an HLA-A03:01 allele, an HLA-A11:01allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01allele, an HLA-A74:01 allele, and/or an HLA-008:02 allele and anycombination thereof.
 30. The composition of any one of claims 1-29,wherein the at least one polypeptide comprises at least one of thefollowing sequences: LVVVGACGV, KLVVVGACGV, LVVVGADGV, KLVVVGADGV,LVVVGAVGV, KLVVVGAVGV, VVGACGVGK, VVVGACGVGK, VVGADGVGK, VVVGADGVGK,VVGAVGVGK, VVVGAVGVGK, VVGACGVGK, VVGADGVGK, VVVGADGVGK, VVGAVGVGK, andVVVGAVGVGK.
 31. The composition of any one of claims 1-30, wherein themutant RAS peptide sequences comprise at least one or two of thefollowing sequences: KLVVVGACGV, FLVVVGACGL, FMVVVGACGI, FLVVVGACGI,FMVVVGACGV, FLVVVGACGV, MLVVVGACGV, FMVVVGACGL, YLVVVGACGV, KMVVVGACGV,YMVVVGACGV, and MMVVVGACGV.
 32. The composition of any one of claims1-31, wherein the mutant RAS peptide sequences comprise at least one ortwo of the following sequences: (a) TEYKLVVVGAVGV;(b) WQAGILARKLVVVGAVGVQGQNLKYQ; (c) HSYTTAEKLVVVGAVGVILGVLLLI;(d) PLTEEKIKKLVVVGAVGVEKEGKISK; (e) GALHFKPGSRKLVVVGAVGVAASDFIFLVT;(f) RRANKDATAEKLVVVGAVGVKELKQVASPF; (g) KAFISHEEKRKLVVVGAVGVKKKLINEKKE;(h) TDLSSRFSKSKLVVVGAVGVKKCDISLQFF;(i) FDLGGGTFDVKLVVVGAVGVKSTAGDTHLG; or(j) CLLLHYSVSKKLVVVGAVGVATFYVAVTVP.


33. The composition of any one of claims 1-32, wherein (Xaa)_(N)comprises an amino acid sequence of IDIIMKIRNA,FFFFFFFFFFFFFFFFFFFFIIFFIFFWMC, FFFFFFFFFFFFFFFFFFFFFFFFAAFWFW,IFFIFFIIFFFFFFFFFFFFIIIIIIIWEC, FIFFFIIFFFFFIFFFFFIFIIIIIIFWEC, TEY,WQAGILAR, HSYTTAE, PLTEEKIK, GALHFKPGSR, RRANKDATAE, KAFISHEEKR,TDLSSRFSKS, FDLGGGTFDV, CLLLHYSVSK, or MTEYKLVVV.
 34. The composition ofany one of claims 1-33, wherein (Xaa_(C))_(C) comprises an amino acidsequence of KKNKKDDIKD, AGNDDDDDDDDDDDDDDDDDKKDKDDDDDD,AGNKKKKKKKNNNNNNNNNNNNNNNNNNNN,AGRDDDDDDDDDDDDDDDDDDDDDDDDDDD, GKSALTIQL, GKSALTI,QGQNLKYQ, ILGVLLLI, EKEGKISK, AASDFIFLVT,KELKQVASPF, KKKLINEKKE, KKCDISLQFF, KSTAGDTHLG,ATFYVAVTVP, LTIQLIQNHFVDEYDPTIEDSYRKQVVIDG, orTIQLIQNHFVDEYDPTIEDSYRKQVVIDGE.


35. The composition of any one of claims 1-34, wherein a first mutantRAS peptide sequence comprises a first neoepitope of a mutant RASprotein and a second mutant RAS peptide sequence comprises a secondneoepitope of a mutant RAS protein, wherein the first mutant RAS peptidesequence is different from the mutant RAS peptide sequence, and whereinthe first neoepitope comprises at least one mutant amino acid and thesecond neoepitope comprises the same mutant amino acid.
 36. Thecomposition of any one of claims 1-35, wherein at least one of themutant RAS peptide sequences comprises a mutant amino acid not encodedby the genome of a cancer cell of a subject.
 37. The composition of anyone of claims 1-36, wherein each of the mutant RAS peptide sequences arepresent at a concentration at least 1 μg/mL, at least 10 μg/mL, at least25 μg/mL, at least 50 μg/mL, or at least 100 μg/mL.
 38. The compositionof any one of claims 1-36, wherein each of the mutant RAS peptidesequences are present at a concentration at most 5000 μg/mL, at most2500 μg/mL, at most 1000 μg/mL, at most 750 μg/mL, at most 500 μg/mL, atmost 400 μg/mL, or at most 300 μg/mL.
 39. The composition of any one ofclaims 1-36, wherein each of the mutant RAS peptide sequences arepresent at a concentration of from 10 μg/mL to 5000 μg/mL, 10 μg/mL to4000 μg/mL, 10 μg/mL to 3000 μg/mL, 10 μg/mL to 2000 μg/mL, 10 μg/mL to1000 μg/mL, 25 μg/mL to 500 μg/mL, or 50 μg/mL to 300 μg/mL.
 40. Thecomposition of any one of claims 1-39, wherein the composition furthercomprises different mutant RAS peptide sequence with a G13A, G13C, G13D,G13R, G13S, G13V, G12A, G12C, G12D, G12R, G12S, G12V or a Q61 mutation.41. The composition of any one of claims 1-40, wherein the compositionfurther comprises an immunomodulatory agent or an adjuvant.
 42. Thecomposition of claim 41, wherein the adjuvant is polyICLC.
 43. Apharmaceutical composition comprising: (a) the composition of any one ofclaims 1-42, and (b) a pharmaceutically acceptable excipient.
 44. Thepharmaceutical composition of claim 43, wherein the pharmaceuticalcomposition comprises a pH modifier present at a concentration of lessthan 1 mM or greater than 1 mM.
 45. The pharmaceutical composition ofclaim 43 or 44, wherein the pharmaceutical composition is a vaccinecomposition.
 46. The pharmaceutical composition of any one of claims43-45, wherein the pharmaceutical composition is aqueous.
 47. Thepharmaceutical composition of any one of claims 43-46, wherein one ormore of the at least one polypeptide is bounded by (a) pI>5 andHYDRO>−6, (b) pI>8 and HYDRO>−8, (c) pI<5 and HYDRO>−5, (d) pI>9 andHYDRO<−8, (e) pI>7 and a HYDRO value of >−5.5, (f) pI<4.3 and−4≥HYDRO≥−8, (g) pI>0 and HYDRO<−8, pI>0 and HYDRO>−4, or pI>4.3 and−4≥HYDRO≥−8, (h) pI>0 and HYDRO>−4, or pI>4.3 and HYDRO≤−4, (i) pI>0 andHYDRO>−4, or pI>4.3 and −4≥HYDRO≥−9, (j) 5≥pI≥12 and −4≥HYDRO≥−9. 48.The pharmaceutical composition of any one of claims 43-47, wherein thepH modifier is a base.
 49. The pharmaceutical composition of any one ofclaims 43-48, wherein the pH modifier is a conjugate base of a weakacid.
 50. The pharmaceutical composition of any one of claims 43-49,wherein the pH modifier is a pharmaceutically acceptable salt.
 51. Thepharmaceutical composition of any one of claims 43-50, wherein the pHmodifier is a dicarboxylate or tricarboxylate salt.
 52. Thepharmaceutical composition of any one of claims 43-50, wherein the pHmodifier is citric acid and/or a citrate salt.
 53. The pharmaceuticalcomposition of claim 52, wherein the citrate salt is disodium citrateand/or trisodium citrate.
 54. The pharmaceutical composition of any oneof claims 43-50, wherein the pH modifier is succinic acid and/or asuccinate salt.
 55. The pharmaceutical composition of claim 54, whereinthe succinate salt is a disodium succinate and/or a monosodiumsuccinate.
 56. The pharmaceutical composition of claim 55, wherein thesuccinate salt is disodium succinate hexahydrate.
 57. The pharmaceuticalcomposition of any one of claims 43-55, wherein the pH modifier ispresent at a concentration of from 0.1 mM-1 mM.
 58. The pharmaceuticalcomposition of any one of claims 43-57, wherein the pharmaceuticallyacceptable carrier comprises a liquid.
 59. The pharmaceuticalcomposition of any one of claims 43-58, wherein the pharmaceuticallyacceptable carrier comprises water.
 60. The pharmaceutical compositionof any one of claims 43-59, wherein the pharmaceutically acceptablecarrier comprises a sugar.
 61. The pharmaceutical composition of claim60, wherein the sugar comprises dextrose.
 62. The pharmaceuticalcomposition of claim 61, wherein the dextrose is present at aconcentration of from 1-10% w/v.
 63. The pharmaceutical composition ofany one of claims 60-62, wherein the sugar comprises trehalose.
 64. Thepharmaceutical composition of any one of claims 60-63, wherein the sugarcomprises sucrose.
 65. The pharmaceutical composition of any one ofclaims 43-64, wherein the pharmaceutically acceptable carrier comprisesdimethyl sulfoxide (DMSO).
 66. The pharmaceutical composition of claim65, wherein the DMSO is present at a concentration from 0.1% to 10%,0.5% to 5%, or 1% to 3%.
 67. The pharmaceutical composition of any oneof claims 43-64, wherein the pharmaceutically acceptable carrier doesnot comprise dimethyl sulfoxide (DMSO).
 68. The pharmaceuticalcomposition of any one of claims 43-67, wherein the pharmaceuticalcomposition is lyophilizable.
 69. The pharmaceutical composition of anyone of claims 43-68, wherein the pharmaceutical composition furthercomprises an immunomodulator or adjuvant.
 70. The pharmaceuticalcomposition of claim 69, wherein the immunomodulator or adjuvant isselected from the group consisting of poly-ICLC, 1018 ISS, aluminumsalts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, ARNAX, STINGagonists, dSLIM, GM-CSF, FLT-3L, IC30, IC31, Imiquimod, ImuFact IMP321,IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryllipidA, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector system,PLGA microparticles, resiquimod, SRL172, Virosomes and other Virus-likeparticles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, and Aquila'sQS21 stimulon.
 71. The pharmaceutical composition of claim 69 or 70,wherein the immunomodulator or adjuvant comprises poly-ICLC.
 72. Thepharmaceutical composition of claim 71, wherein a ratio of poly-ICLC topeptides in the pharmaceutical composition is from 2:1 to 1:10 v:v. 73.The pharmaceutical composition of claim 72, wherein the ratio ofpoly-ICLC to peptides in the pharmaceutical composition is about 1:1,1:2, 1:3, 1:4 or 1:5 v:v.
 74. The pharmaceutical composition of claim73, wherein the ratio of poly-ICLC to peptides in the pharmaceuticalcomposition is about 1:3 v:v.
 75. A method of treating a subject withcancer comprising administering to the subject the pharmaceuticalcomposition of any one of claims 43-74.
 76. A method of treating asubject with cancer comprising administering to the subject a peptidewith a sequence of VVGADGVGK, VVGACGVGK, VVGAVGVGK, VVVGADGVGK,VVVGACGVGK, VVVGAVGVGK, wherein the subject expresses a protein encodedby an HLA-A02:01 allele, an HLA-A03:01 allele, an HLA-A11:01 allele, anHLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, anHLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, anHLA-A74:01 allele, or an HLA-008:02 allele of the subject's genome. 77.A method of treating a subject with cancer comprising administering tothe subject a mutant RAS peptide or a nucleic acid encoding the mutantRAS peptide, wherein the mutant RAS peptide comprises at least 8contiguous amino acids of a mutant RAS protein comprising a mutation atG12, wherein the peptide comprises the mutation at G12 and binds toHLA-A11:01 or HLA-A03:01, wherein the subject is identified asexpressing a protein encoded by an HLA-A03:01 allele, an HLA-A11:01allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01allele, or an HLA-A74:01 allele.
 78. A method of treating a subject withcancer comprising administering to the subject a peptide comprising asequence GADGVGKSAL, GACGVGKSAL, GAVGVGKSAL, GADGVGKSA, GACGVGKSA, orGAVGVGKSA; wherein the subject expresses a protein encoded by anHLA-A02:01 allele, an HLA-A03:01 allele, an HLA-A11:01 allele, anHLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, anHLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, anHLA-A74:01 allele, or an HLA-008:02 allele of the subject's genome thatbinds to the peptide.
 79. A method of treating a subject with cancercomprising administering to the subject a first and a second peptide ora nucleic acid encoding the first and second peptide, wherein the firstand second peptides comprise at least two of: (1) KLVVVGADGV,KLVVVGACGV, KLVVVGAVGV, LVVVGADGV, LVVVGACGV, LVVVGAVGV; (2) GADGVGKSAL,GACGVGKSAL, GAVGVGKSAL, GADGVGKSA, GACGVGKSA, GAVGVGKSA; and (3)VVGADGVGK, VVGACGVGK, VVGAVGVGK, VVVGADGVGK, VVVGACGVGK, VVVGAVGVGK;wherein the subject's HLA allele expression is unknown at the time ofadministration.
 80. A method of treating a subject with cancercomprising administering to the subject a mutant RAS peptide or anucleic acid encoding the mutant RAS peptide, wherein the mutant RASpeptide comprises at least 8 contiguous amino acids of a mutant RASprotein comprising a G12C mutation, wherein the peptide comprises theG12C mutation, and further wherein the peptide comprises a stabilizingmutation not encoded by a genome of a cancer cell, wherein the subjectexpresses a protein encoded by an HLA-A02:01 allele, an HLA-A03:01allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03allele, an HLA-A68:01 allele, an HLA-A74:01 allele, or an HLA-008:02allele.
 81. A method of identifying a subject with cancer as a candidatefor a therapeutic, the method comprising identifying the subject as asubject that expresses a protein encoded by an HLA-A03:01 allele, anHLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, anHLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, anHLA-A68:01 allele, or an HLA-A74:01 allele, wherein the therapeutic is amutant RAS peptide or a nucleic acid encoding the mutant RAS peptide,wherein the mutant RAS peptide comprises at least 8 contiguous aminoacids of a mutant RAS protein comprising a mutation at G12, wherein thepeptide comprises the mutation at G12 and binds to a protein encoded byan HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, anHLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, anHLA-A33:03 allele, an HLA-A68:01 allele, or an HLA-A74:01 allele. 82.The method of claim 81, wherein the method further comprisesadministering the therapeutic to the subject.
 83. A method of treating asubject with cancer, the method comprising: (a) identifying a firstprotein expressed by the subject, wherein the first protein is encodedby a first HLA allele of the subject and wherein the first HLA allele isan HLA allele provided in any one of Tables 1 to 14; and (b)administering to the subject (i) a first mutant RAS peptide, wherein thefirst mutant RAS peptide is a peptide to the first HLA allele providedin any one of Tables 1 to 14, or (ii) a polynucleic acid encoding thefirst mutant RAS peptide.
 84. The method of claim 83, further comprisingidentifying a second protein expressed by the subject, wherein thesecond protein is encoded by a second HLA allele of the subject andwherein the second HLA allele is an HLA allele provided in any one ofTables 1 to
 14. 85. The method of claim 84, further comprisingadministering to the subject (i) a second mutant RAS peptide, whereinthe second mutant RAS peptide is a peptide to the second HLA alleleprovided in any one of Tables 1 to 14, or (ii) a polynucleic acidencoding the second mutant RAS peptide.
 86. The method of claim 84 or85, wherein the first HLA allele is different from the second HLAallele.
 87. The method of claim 84 or 85, wherein the first mutant RASpeptide is different from the second mutant RAS peptide.
 88. The methodof any one of claims 75-87, wherein an immune response is elicited inthe subject.
 89. The method of claim 88, wherein the immune response isa humoral response.
 90. The method of any one of claims 75-89, whereinthe mutant RAS peptide sequences are administered simultaneously,separately or sequentially.
 91. The method of claim 90, wherein thefirst peptide is sequentially administered after a time periodsufficient for the second peptide to activate the second T cells. 92.The method of any one of claims 75-91, wherein the cancer is selectedfrom the group consisting of lung cancer, non-small cell lung cancer,pancreatic cancer, colorectal cancer, uterine cancer and liver cancer.93. The method of any one of claims 75-92, further comprisingadministering at least one additional therapeutic agent or modality. 94.The method of claim 93, wherein the at least one additional therapeuticagent or modality is surgery, a checkpoint inhibitor, an antibody orfragment thereof, a chemotherapeutic agent, radiation, a vaccine, asmall molecule, a T cell, a vector, and APC, a polynucleotide, anoncolytic virus or any combination thereof.
 95. The method of claim 94,wherein the at least one additional therapeutic agent is an anti-PD-1agent and anti-PD-L1 agent, an anti-CTLA-4 agent, or an anti-CD40 agent.96. The method of claim 94 or 95, wherein the additional therapeuticagent is administered before, simultaneously, or after administering themutant RAS peptide sequences.